Cationic Antimicrobial Peptides: Thermodynamic ...

Cationic Antimicrobial Peptides: Thermodynamic

Characterization of Peptide-Lipid Interactions and

Biological Efficacy of Surface-Tethered Peptides

Dissertation zur Erlangung des akademischen Grades des

Doktors der Naturwissenschaften (Dr. rer. nat.)

eingereicht im Fachbereich Biologie, Chemie, Pharmazie

der Freien Universität Berlin

vorgelegt von

Mojtaba Bagheri

aus Marvdasht (Iran)

Juni 2010

Hereby, I declare that I have prepared this work independently under supervision of

Dr. Margitta Dathe in the time period from October 2006 until April 2010 at Leibniz-Institut

für Molekulare Pharmakologie (FMP) in Berlin. To best of my knowledge, this thesis contains

no previously published materials by another person.

1. Gutachterin: Prof. Beate Koksch, Freie Universität Berlin

2. Gutachter: Prof. Michael Bienert, Leibniz-Institut für Molekulare Pharmakologie

Disputation am 09 September 2010

To my parents

Acknowledgments

I would like to show my appreciation to Prof. Dr. Michael Bienert (FMP), the head of

Department of Chemical Biology of FMP, and my PhD supervisors Dr. Margitta Dathe

(FMP) and Prof. Dr. Beate Koksch (Freie Universität Berlin) for giving me the opportunity to

work in the interesting field of antimicrobial peptides and their support during my doctoral

thesis research.

I would especially like to thank Dr. Michael Beyermann (FMP), and

Prof. Dr. Sandro Keller (University of Kaiserslautern) for their informative discussions and

suggestions.

Furthermore, Mrs. Annerose Klose, Mrs. Angelika Ehrlich, Mrs. Dagmar Krause, and

Mr. Bernhard Schmikale are thanked for their assistance in the field of peptide synthesis,

HPLC and mass measurements. I would also like to acknowledge Mr. Rudolf Dölling

(Biosyntan GmbH) for the synthesis of c-(1MeW)F(1MeW), c-(5MeoW)F(5MeoW), and c-

(5fW)F(5fW).

I would also like to thank Mrs. Heike Nikolenko and Christof Junkes for their support

in maintaining cell cultures. I am very grateful for the generous technical support of

Nadin Jahnke, Gerdi Kemmer, and Katharina Grimm (all FMP) with ITC and CD expriments.

Special thanks to Ewan St. John Smith (Max Delbrück Center for Molecular

Medicine) and Gesa Schäfer (FMP) for the time they spent on correcting my thesis as well as

for their fruitful suggestions.

Finally, I would like to thank my parents for their understanding and patience and their

spiritual support in my life.

Content

Content

ABBREVIATIONS AND SYMBOLS .....................................................................................I

Abbreviations ......................................................................................................................................................... I

Symbols ..................................................................................................................................................................V

1 INTRODUCTION................................................................................................................. 1

1.1 CAPs................................................................................................................................................................. 1 1.1.1 Structure diversity and basis of activity..................................................................................................... 1

1.2 Cellular basis of activity and bacterial selectivity ........................................................................................ 4 1.2.1 Membrane composition ............................................................................................................................. 4 1.2.2 Bacterial LPS............................................................................................................................................. 6

1.3 Mechanims of action ....................................................................................................................................... 9 1.3.1 Membrane permeabilization ...................................................................................................................... 9 1.3.2 Alternative mechanisms of action............................................................................................................ 11

1.4 Small CAPs .................................................................................................................................................... 11 1.4.1 Significance of short CAPs...................................................................................................................... 11 1.4.2 Particular properties of RW-rich hexapeptides ........................................................................................ 13

1.5 Surface-tethered peptides ............................................................................................................................. 14 1.5.1 Inhibition of biofilm formation................................................................................................................ 14 1.5.2 Peptide-based biofilm .............................................................................................................................. 18

2 AIMS OF THE STUDY...................................................................................................... 20

2.1 Structural basis of anti-E. coli activity of cyclic RW-rich hexapeptides .................................................. 20

2.2 Preparation and perspectives of surface-tethered peptides....................................................................... 21

3 RESULTS AND DISCUSSION.......................................................................................... 23

3.1 W-substituted c-WFW analogs .................................................................................................................... 23 3.1.1 Description and physicochemical properties of W-analogs..................................................................... 23 3.1.2 Cyclic peptide synthesis and their HPLC characterization ...................................................................... 25 3.1.3 Characterization of cyclic peptides by CD .............................................................................................. 26 3.1.4 Antibacterial and hemolytic activities ..................................................................................................... 29 3.1.5 Cyclic peptide binding to lipid bilayers determined by ITC.................................................................... 31

3.1.5.1 Lipid bilayers as model of biological membranes ............................................................................ 31 3.1.5.2 Peptide accumulation as reflected by apparent binding.................................................................... 32

Influence of lipid composition upon binding ........................................................................................... 32 Role of sequence composition upon binding ........................................................................................... 34

3.1.5.3 Peptide partitioning in lipid bilayers ................................................................................................ 37 Influence of lipid composition upon binding ........................................................................................... 37 Role of sequence composition upon binding ........................................................................................... 38

3.1.5.4 Effect of ionic strength upon c-WFW binding to r-LPS and s-LPS lipid systems ........................... 42

Content

3.1.5.5 Heat capacity change on membrane partitioning of c-WFW............................................................ 44 3.1.6 Summary.................................................................................................................................................. 46

3.2 Site-specific immobilization of CAPs........................................................................................................... 49 3.2.1 Physical and chemical properties of PEGylated resins as model solid surfaces ...................................... 50 3.2.2. Activity of surface tethered membrane-active CAPs - role of tethered peptide site ............................... 51

3.2.2.1 Characterization of KLAL and MK5E peptides by HPLC and CD.................................................. 51 3.2.2.2 Preparation and characterization of tethered KLAL and MK5E peptides ........................................ 53 3.2.2.3 Biological activities of free and tethered KLAL and MK5E peptides.............................................. 55

Antimicrobial activity of the free peptides............................................................................................... 55 Antimicrobial activity of the tethered peptides ........................................................................................ 57 Hemolytic activity of the free and tethered peptides................................................................................ 59

3.2.2.4 Bilayer permeabilizing activities of free and tethered KLAL and MK5E peptides.......................... 59 The free peptides...................................................................................................................................... 60 The tethered peptides ............................................................................................................................... 61

3.2.3 Influence of physical characteristics of solid surfaces upon biocidal activity ......................................... 62 3.2.3.1 Effect of spacer length...................................................................................................................... 62 3.2.3.2 Surface density of tethered peptides................................................................................................. 62 3.2.3.3 Effect of particle size........................................................................................................................ 63

3.2.4 Peptide-tethering as a strategy to investigate the mode of action of CAPs.............................................. 65 3.2.4.1 Characterization of MEL, BUF, and TP peptides............................................................................. 65 3.2.4.2 Characterization of tethered MEL, BUF, and TP peptides............................................................... 67 3.2.4.3 Biological activities of free and tethered peptides............................................................................ 67

Antimicrobial activity of free peptides .................................................................................................... 67 Inner and outer membrane-permeabilizing activities of MEL, BUF, and TP peptides ............................ 69 Antimicrobial activity of tethered peptides.............................................................................................. 70

3.2.4.4 Bilayer permeabilizing activities of free and tethered MEL, BUF, and TP peptides ....................... 71 3.2.5 Summary.................................................................................................................................................. 73

4 SUMMARY.......................................................................................................................... 76

4.1 W-substituted c-WFW analogs .................................................................................................................... 76

4.2 Site-specific immobilization of CAPs........................................................................................................... 78

5 ZUSAMMENFASSUNG .................................................................................................... 80

5.1 W-substituierten c-WFW Analoga .............................................................................................................. 80

5.2 Ortsspezifische Immobilisierung von CAPs................................................................................................ 82

6 EXPERIMENTAL SECTION ........................................................................................... 84

6.1 Materials: Chemicals and reagents ............................................................................................................. 84

6.2 Methods.......................................................................................................................................................... 84 6.2.1 Synthesis of peptides ............................................................................................................................... 84

6.2.1.1 Synthesis of linear peptides (automated synthesis) .......................................................................... 84 6.2.1.2 Synthesis of cyclic peptides (manuel synthesis)............................................................................... 85 6.2.1.3 HPLC purification of crude peptides................................................................................................ 86 6.2.1.4 Characterization of peptides based upon tR-HPLC........................................................................... 86

6.2.2 Immobilization of CAPs .......................................................................................................................... 87

Content

6.2.2.1 SPPS: C terminus immobilization .................................................................................................... 87 6.2.2.2 Thioalkylation: N terminus and side chain immobilization of KLAL.............................................. 87 6.2.2.3 Oxime-forming ligation: N terminus and side chain immobilization of MK5E, MEL, BUF, and TP...................................................................................................................................................................... 87 6.2.2.4 Characterization of tethered peptides using UV-absorption of the Fmoc-chromophore .................. 88

6.2.3 Antimicrobial activity .............................................................................................................................. 88 6.2.3.1 Bacterial culture preparation and determination of MIC and MBC ................................................. 88

6.2.4 Hemolytic activity ................................................................................................................................... 90 6.2.5 Inner and outer membrane permeabilizing activities of MEL, BUF and TP and their AOA-modified analogs.............................................................................................................................................................. 91 6.2.6 Vesicle preparation .................................................................................................................................. 92

6.2.6.1 SUVs ................................................................................................................................................ 92 Synthetic lipids ........................................................................................................................................ 92 Natural lipids............................................................................................................................................ 92

6.2.6.2 LUVs ................................................................................................................................................ 93 6.2.7 Lipid bilayer permeabizing activities....................................................................................................... 94 6.2.8 Isothermal titration calorimetry ............................................................................................................... 95

6.2.8.1 Theory and description of surface partitioning equilibrium model .................................................. 96 6.2.8.2 Instrument setup and measurement .................................................................................................. 98 6.2.8.3 ITC data analysis and curve fitting................................................................................................... 99

6.2.9 CD spectroscopy...................................................................................................................................... 99 6.2.9.1 c-WFW and W-subtituted analogs ................................................................................................. 100 6.2.9.2 Model KLAL peptide, MK5E, and the PEGylated analogs............................................................ 100

7 REFERENCES .................................................................................................................. 101

8 APPENDIX ........................................................................................................................ 119

8.1 Curriculum vitae ......................................................................................................................................... 119

8.2 Publications and scientific conference contributions ............................................................................... 119 8.2.1 Original publications ............................................................................................................................. 119 8.2.2 Scientific conference contributions........................................................................................................ 120

Abbreviations and Symbols

I


Abbreviations 1MeW 1-Methyl-L-tryptophan

1-PrOH 1-Propanol

5fW 5-Fluoro-L-tryptophan

5MeoW 5-Methoxy-L-tryptophan

5MeW 5-Methyl-DL-tryptophan

ACN Acetonitrile

Ac2O Acetic anhydride

Ac-RW Ac-RRWWRF-NH2

AOA Aminooxy acetic acid

Arg, R Arginine

Asn, N Asparagine

Asp, P Aspartic acid

b3-hW L--Homotryptophan

Bal -(Benzothien-3-yl)-L-alanine

Boc tert-Butyloxycarbonyl

BrAcOH Bromoacetic acid

BSA Bovine serum albumin

BUF Buforin 2

CAP Cationic antimicrobial peptide

CL Cardiolipin

CD Circular dichroism

ClTrt-Cl (2'-Chloro)-chlorotrityl polystyrene

c-RW cyclo-RRWWRF

c-WFW cyclo-RRRWFW

Cys, C Cysteine

DCM Dichloromethane

Dde 4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene ethyl

Dht (S/R)-Dihydrotryptophan

DLS Dynamic light scattering


II

DIC N',N'-Diisopropylcarbodiimide

DIEA N',N'-diisopropylethylamine

DMF Dimethylformamide

DPPC 1,2-Dipalmitoyl-sn-glycero-3-phospho-choline

DPPE 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine

DPPG 1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

DVB Divinylbenzene

EC25 Quarter maximal effective concentration

EC50 Half maximal effective concentration

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDT 1,2-Ethandithiol

Fmoc 9-Fluorenylmethoxycarbonyl

Fmoc-Cl Chloroformic acid 9-fluorenylmethyl ester

Gal Galactose

GalNAc N-Acetyl-galactosamine

Glc Glucose

GlcN Glucosamine

GlcNAc N-Acetyl-glucosamine

Gln, Q Glutamine

Glu, E Glutamic acid

Gly, G Glycine

GnHCl Guanidinium hydrochlorid

HAPyU 1-(1-Pyrrolidinyl-1H-1,2,3-triazolo [4,5-b] pyridine-1-ylmethylene) pyrrolidinium hexafluorophosphate N-oxide

HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate

Hep L-Glycerol-D-manno-heptose

His, H Histidine

HMPA 4-(Hydroxymethyl)phenoxyacetic acid

HOBt Hydroxybenzotriazole

HOEGMA Hydroxyl-terminated oligo(ethyleneglycol) methacrylate

Igl (S)-(2-Indanyl)glycine

ITC Isothermal titration calorimetry


III

Kdo 3-deoxy-D-manno-oct-2-ulosonic acid

LA Lipid A

LB Luria broth

LC/ESI-TOF MS liquid chromatography/electrospray ionization time-of-flight mass spectrometry

LPS Lipopolysaccharide

LUV Large unilamellar vesicles

Lys, K Lysine

MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry

MBC Minimal bacteriocidal concentration

MBHA 4-Methylbenzhydrylamine hydrochloride

MEO2MA Poly(2-(2-methoxyethoxy)ethyl methacrylate

MEL Melittin

MG 1 Magainin 1

MG 2 Magainin 2

MIC Minimal inhibitory concentration

Nal -(2-naphthyl)alanine

NCF Nitrocefin

NHS N-hydroxysuccinimide

NMI N-Methylimidazole

NMR Nuclear magnetic resonance

O.D. Optical density

ONPG o-nitrophenyl--D-galactopyranoside

PAA Polyacrylic acid

Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PEG 2 8-amino-3,6-dioxaoctanoic acid

PEG Polyethylene glycol

Phe, F Phenylalanine

PMPI N-(p-maleimidophenyl)isocyanate

POPC 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline

POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG 1-Palmitoyl-2-oleoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]


IV

POPI 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol

POPS 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylserine

Pro, P Proline

PVP Polyvinylpyrrolidone

RBC Red blood cell

r-LPS Rd-lipopolysaccharide

RP-HPLC Reversed-phase high performance liquid chromatography

RW RRWWRF

SAR Structure-activity relationship

SDS Sodium lauryl sulfate

Ser, S Serine

s-LPS Smooth-lipopolysaccharide

SM Sphingomyelin

SPPS Solid-phase peptide synthesis

SUV Small unilamellar vesicles

TBP Tributyl phosphine

t-Bu tert-butyl

TFA Trifluoroacetic acid

TFE 2,2,2-trifluoroethanol

Thr, T Threonine

TIPS Triisopropylsilane

TP Tritrpticin

tR Retention time

TRIS Tris(hydroxymethyl) aminomethane

Trt Trityl

Tyr, Y Tyrosine

UV−VIS Ultraviolet−Visible


V

Symbols AL Membrane area occupied by one lipid headgroup

AP Membrane area occupied by the peptide

cL Total lipid concentration

cP,b Concentration of membrane surface-bound peptide

cP,f Bulk concentration of peptide

cP,i Interfacial concentration of peptide

Cp° Molar heat capacity changes

G° Standard Gibbs free energy of membrane partitioning

H° Standard molar enthalpy of membrane partitioning

S° Standard molar entropy of membrane partitioning

0 Electric permittivity of free space

r Dielectric constant of water

F0 Faraday constant

Lipid accessibility factor

K0 Intrinsic partition coefficient

Kapp. Apparent binding constant

Wavelength

mr Mean residue molar ellipticity

R Gas constant

Rb molar ratio of bound peptide over accessible lipid

Membrane surface charge density

Vcell Volume of the calorimeter cell

Membrane surface potential

zP Effective charge number of peptide

Introduction

1

1 Introduction

1.1 CAPs

CAPs have been found in almost all species of eukaryotic organisms and are

recognized as the evolutionarily conserved components of their innate immune system that

defend the host against microbes through membrane or metabolic disruption [1]. A

distinguishable advantage of CAPs over conventional antibiotics is that they do not provoke

immune responses [2]. Regardless of their origin and biological efficacy, CAPs share

common features. They are short peptides (usually between 350 amino acid residues), which

include a large content of basic amino acids and a global distribution of hydrophobic and

hydrophilic residues. Due to the high frequency of amino acid residues, such as R and K in

their sequences, they carry an overall positive charge in the physiological pH range, which is

of great importance for their interactions with the negative charges of bacterial cell

membranes [1]. CAPs adapt an amphipathic conformation at polar-nonpolar interfaces with a

hydrophobic domain consisting of nonpolar amino acid residues on one side and polar or

charged residues on the opposite (Fig. 1). Because of these physicochemical characteristics,

CAPs have a tendency to accumulate on the negatively charged microbial surfaces and

membranes. Each CAP has a unique pattern of activity against a variety of Gram-positive and

Gram-negative bacteria, yeasts, fungi and viruses [4]. While the antimicrobial peptide

cecropin A is only active against Gram-positive bacteria [5], MG 2 and dermaseptin show

activity against both types of bacteria as well as fungi [6,7], and the membrane-lytic peptide

MEL attacks both prokaryotic and mammalian cells [8].

1.1.1 Structure diversity and basis of activity

In spite of their common mechanism of action, CAPs show remarkable structural

diversity. The relationship between their structure and the spectrum of antimicrobial activity

is a complex subject, because even CAPs of the same structure may have different effects

upon microorganisms and mammalian cells [9,10]. Due to the high structural diversity of the

large number of peptides discovered so far, it is difficult to classify CAPs into generally

accepted groups. Nevertheless, CAPs can be roughly classified based upon their secondary

structure into four major categories: -helical, -sheet, loop, and extended peptides [11]

(Fig. 2). CAPs with -helical and -sheet secondary structures are among the most ubiquitous

Introduction

2

peptides in nature. Besides this classification, there are a few groups of CAPs, which are

classified based on frequent occurrence of one or more amino acid residues, e.g., Bac5 [13]

and PR-39 [14] are PR-rich antimicrobial peptides; indolicidin has a high content of

W residues, and histatin 5 [15] contains numbers of H residues.

Fig. 1. Helical wheel projection of the amphipathic -helix in alamethicin, MG 2, and model KLAL peptide. The positively charged residues are presented in black, the hyrdrophilic residues in gray, and the

hydrophobic residues in white colors. Q, H, , and represent the total positive charge of the peptide, peptide hydrophobicity, the hydrophobic moment, and the angle subtended by cationic residues, respectively. Adapted

from ref. [3].

Introduction

3

Fig. 2. Schematic description of the models representing the structure of CAPs. The strutures from top to bottom are: (a) -sheet structure; e.g., human -defensin 1, which forms a triple--sheet structure stabilized by three disulfide bonds. The N-terminal region of -defensin 1 also contributes to an -helix segment. (b) -helix structure, e.g., MG 2, (c) extended structure, e.g., indolicidin, which does not contain structural elements of -helices or -sheets. Instead, it has a stretched spiral structure; (d) loop, which is restrained by disulfide bridge,

e.g., tachyplesin. It adopts a -hairpin fold. Taken from ref. [12].

The knowledge and understanding of activity-modulating structural parameters

responsible for the antibacterial activity and specificity of CAPs may provide insight into both

the relationship between structure and activity as well as the mechanism of action. Because of

their wide dissemination, -helical peptides are the best-studied CAPs. Dathe et al.

demonstrated that the physicochemical parameters of CAPs, such as (i) the helicity and

amphipathicity, (ii) the hydrophobicity, (iii) the hydrophobic moment, (iv) the magnitude of

the charge, and (v) the angle subtended by the charged helix face are effective modulators for

biological activity and membrane selectivity of the peptides (Fig. 1) [16]. Using an approach

of minimal peptide sequence modification, Wieprecht and Dathe et al. studied the influence of

these structural parametes upon membrane activity for MG 2-analog peptides [17−20]. They

showed that an enhanced hydrophobic moment or increased hydrophobicity resulted in more

potent antimicrobial peptides. In contrast, the disturbance of the amphipathic helix due to

double D-amino acid substitution, particularly in the stable helix region of the peptides, led to

less membrane activity. In addition, an enlarged cationic domain increased lipid affinity and

Introduction

4

permeabilizing activity of the peptides. The reinforcement of the electrostatic interactions

between positively charged peptides and negatively charged membrane components had a

higher influence upon the bactericidal acivity than on the hemolytic effect.

1.2 Cellular basis of activity and bacterial selectivity

1.2.1 Membrane composition

The majority of CAPs work differently compared to conventional antibiotics, which

generally block an enzymatic activity essential for a microorganism’s reproduction through a

specific interaction with a protein or a protein-nucleic acid complex [21]. Because CAPs often

act via permeabilization of the bacterial cell membranes; one should consider the differences

in structure of the outer layer of bacterial species, their cell profiles, and the variable

composition of lipids in the plasma membrane in order to understand the basis of their

different spectra of activity.

Bacterial cell membranes are usually rich in anionic lipids [1]. A schematic

representation of the cell wall of Gram-positive and Gram-negative bacteria is shown in

Fig. 3. The cell enevelope of Gram-negative bacteria consists of an outer and an inner

(cytoplasmic) membrane. The outer membrane is highly asymmetric, with the outer leaflet

covered by highly negatively charged LPS ( 90%) and the inner leaflet mainly composed of

POPE. A thin layer of peptidoglycan, which constitutes the largest part of the preplasmic

space, covers the distance between the outer and cytoplasmic membranes. The cytoplasmic

membrane, which is considered as the target for CAPs, is composed of: POPE, negatively

charged POPG phospholipid and cardiolipin. In contrast, Gram-positive bacteria have no

outer membrane, but a thick peptidoglycan layer which protects the cytoplasmic membrane.

The main difference arises from the ratio of POPE and POPG phospholipids in the

cytoplasmic membrane of Gram-positive and Gram-negative bacteria (Table 1). Whereas the

content of negatively charged lipids is higher in Gram-positive bacteria (approximately

60−90% of the total phospholipids), a much higher proportion of POPE is found in the inner

membrane of Gram-negative strains (Table 1) [22]. The difference in the molar ratio of POPG

and POPE may be important for the lateral organization, lipid packing and/or mobility, which

can be modified in the interaction with other moieties in particular with CAPs.

Introduction

5

Fig. 3. Schematic representation of the cell wall structure of Gram-positive and Gram-negative bacteria.

Table 1. Phospholipid composition of the plasma membranes of selected microorganisms. Adapted from ref. [22,23].

Phospholipid Composition (%)Wt Species/cell POPG POPS POPC POPE CL Other

Cholesterol

LPS

Fungi

C. albicans 0 11 4 70 0 15* −† −

C. neoformans 0 16 51 29 4 0 − −

Bacteria

Gram-positive

S. aureus 57 0 0 0 5 38‡ − −

S. epidermis 90 0 0 0 1 9 − −

B. megaterium 40 0 0 40 5 15 − −

B. subtilis 70 0 0 12 4 12 − −

Gram-negative

E. coli 15 0 0 80 5 0 − +

S. typhimurium 33 0 0 60 7 0 − +

B. cepacia 18 0 0 82 0 0 − +

Erythrocytes

RBCs 0 14 31 30 0 25§ + − *Almost exclusively SM

† − ; not present, + ; present

‡Almost exclusively lyso-POPG

§SM: 24%, POPI: 1%

Introduction

6

The bacterial negatively charged outer surface provides the basis for accumulation of cationic

peptides, which is driven by electrostatic interactions. Compared to bacterial membranes, the

outer layer of the mammalian cell membrane contains exclusively zwitterionic POPC, POPE,

and neutral SM, and the inner layer contains POPE and POPS [24]. In addition, the plasma

membrane of mammalian cells is characterized by the presence of cholesterol in both layers.

This endows the membrane with different mechanical properties responsible for reduced

binding of amphipathic molecules, such as CAPs to these membranes (Fig. 4) [25].

Fig. 4. Schematic description of the molecular basis of cell selectivity of CAPs. Taken from ref. [1]

1.2.2 Bacterial LPS

LPS is quite different from the membrane phospholipids and plays an important role

as an efficient barrier to certain hydrophobic antibiotics [26]. As a result of destroying Gram-

negative bacteria, LPS can be released from the cell wall into the blood stream and induce

septic shock [27]. Thus, the interaction of peptides with LPS for both the antibacterial, as well

as the anti-inflammatory, effects of the peptides is of crucial importance. LPS is an

amphiphilic heteropolymer and consists of a conserved fatty acyl chain backbone known as

Introduction

7

LA and a large polysaccharide chain [28]. LA is the toxic moiety of LPS. The backbone of

LA consists of a diglucosaminecarbohydrate backbone in a beta-(1'→6)-linkage, which is

phosphorylated at positions 1 and 4' (Fig. 5) [28]. A total of four 3-hydroxy-fatty acyl chains,

generally having a length of 12 to 14 carbon atoms and known as “fatty acids primary”, are

attached directly to the carbohydrate backbone by either ester- or amide-linkages (Fig. 5). The

primary 3-hydroxy fatty acids can be further esterified by a total of 4−7 fatty acids. These

fatty acid chains are so-called “fatty acids secondary”, which are not directly bound to LA.

Fig. 5. Schematic structure of LPS from Brucella spp. Taken from ref. [29].

The polysaccharide structure of LPS consits of an oligosaccharide core with a limited

number of sugars, and highly variable polysaccharide chains composed of one or more

oligosaccharide repeating units, the so-called O-antigen (Fig. 5). The O-antigen has several

biological activities; it serves as a receptor for bacteriophages, modulates the activation of the

alternative complement pathway, and inhibits the attachment of membrane attack molecules

to the bacterial outer membrane [26,30]. When an LPS contains the O-antigen chain, it is

described as s-form LPS (smooth) because the bacterial colonies have a smooth morphology

Introduction

8

(Fig. 6). However, not all Gram-negative bacteria possess an O-antigen. Such bacteria are

termed rough (r) because they form rigid, incomplete colonies on solid agar and

autoagglutinate in saline. Several rough (r)-mutants were shown to have truncated

polysaccharide cores due to defects in genes that code for glycosyl or phosphoryl transferases.

Depending upon the length of the core region of the polysaccharides, they are described as

Re-, Rd-, Rc-, Rb-, and Ra-LPS (Fig. 6) [28]. The rough mutant containing the complete core

is termed Ra-LPS. LPS which lack the terminal sugar are known as Rb-LPS, and Re-LPS is

the shortest LPS mutant which occurs in nature. Re-LPS contains only two singly-negatively

charged Kdo.

Fig. 6. General structure of E. coli LPS. The diversity of sugar moieties in the core region and the composition of LA are conserved in different E. coli strains. However; the O-antigen region varies strongly among different E. coli strains. Charged residues are located preferentially in the core oligosaccharide region. Ra–Re denote the

different rough chemotypes of LPS. Native, complete LPS is called smooth LPS (s-LPS). Adapted from ref. [28].

LPS possess binding sites for calcium and magnesium ions are responsible for the

structural stability of the outer membrane of Gram-negative bacteria [31]. The susceptibility

of Gram-negative bacteria to CAPs has been proposed to be associated with factors that

facilitate the transport of the peptide across the outer membrane, such as the magnitude and

the location of the LPS charge, the concentration of LPS in the membrane and the absence of

O-antigen chains. CAPs are either trapped in the outer barrier [32] or increase their

permeability by causing disordering of the LPS organization [12]. A mechanism of “self-

promoted uptake” has been proposed for the transport of CAPs across the complex outer

membrane, thus facilitating peptide accessibility to the inner target membrane [12].

Introduction

9

1.3 Mechanims of action

1.3.1 Membrane permeabilization

Most of the soluble CAPs penetrate into the cell membrane and enhance the

permeability. Electrostatic interactions cause the accumulation of the cationic peptides at the

negatively charged bacterial membrane, and hydrophobic interactions drive their insertion

into the lipid bilayer [33]. Additionally, counter ion exchange initiating a “self-promoted

uptake” across the outer LPS–rich membrane modulates the peptide accessibility to the inner

target membrane of Gram-negative bacteria [12]. Several models have been proposed to

explain the modes of action of CAPs, which are presented in Fig. 7. However, the evidence

for the anticipated modes of action is based on experiments carried out on model lipid systems

[34]. Peptides bind to the membrane by assuming an amphipathic helix with the polar domain

exposed to the membrane surface and the hydrophobic helix face buried in the lipid acyl chain

region. Changes in the lipid bilayer structure and reorientation of the bound peptides lead to

membrane permeabilization.

(A) barrel-stave model (transmembrane helix bundles)

This model describes a pore as an aggregated set of membrane-bound CAPs. The axes

of the -helical sequences are oriented perpendicular to the membrane surface (Fig. 7A). The

peptide sequences should be long enough to span a 30 Å thick lipid membrane, and thus this

model is not appropriate for short CAPs. A typical example for this model is alamethicin,

which forms stable ion channels [35]. The pore formation is initiated with alamethicin

N terminus insertion into the membrane interior where the peptide hydrophobic region binds

to the hydrophobic chains of lipids and the peptide hydrophilic region forms a central lumen.

The channel diameters range from 0.2 and 2 nm [36] and the number of helices which form

the alamethicin pore varies between 3−11 [37,38].

(B) toroidal model (wormhole)

The toroidal pore model is comparable to the barrel-stave model; however, the pores

are instable and poorly reproducible (Fig. 7B) [33]. In this model, the peptides associate with

the polar lipid headgroups and penetrate into the membrane. The bound peptides cause lipid

chain disorder and induce an enhanced lipid surface expansion and curvature modulation,

which lead to an unfavorable strain between the outer and inner lipid layers. At high peptide

concentration, the peptides are lined perpendicular to the membrane surface. Then, both

Introduction

10

peptides and lipids form a transmembrane pore which connects the inner and outer lipid layers

[39]. The membrane activity of MG 2 is associated with this type of pore [40−42]. The pore

diameter reaches 3 nm, about twice as large as the alamethicin channel [43].

Fig. 7. The classical models for the mode of membrane permeabilization of CAPs. The black cylinders represent the peptides. (A) barrel-stave model (e.g., alamethicin), (B) toroidal pore model (e.g., MG 2), and (C)

Carpet-like model (e.g., model KLAL peptide). Taken from ref. [3].

(C) carpet-like model

This model reflects the mode of action of short amphiphilic CAPs, which are not able

to span the membrane. Based on this model, membrane permeabiliziaton happens through the

following steps. (i) The peptides electrostatically accumulate at the membrane surface. (ii)

After reaching a threeshold concentration, the peptides orientate parallel to the membrane

surface where their hydrophobic surface points towards the hydrophobic interior of the lipid

bilayer, and their hydrophilic surface is in contact with the phospholipid headgroups. (iii) The

peptides self-associate on the bilayer, leading to high surface tension, destruction of

membrane integrity, and consequently membrane collapse (Fig. 7C) [33]. In this scenario no

Introduction

11

local CAPs bundles will be formed. Model KLAL peptides have been suggested to

permeabilize the lipid bilayer according to this mode of action [44].

1.3.2 Alternative mechanisms of action

In spite of the membrane-permeabilizing activities, a different view on the mechanism

of action of CAPs involves their penetration across the membranes of pathogens without pore

formation [4]. This process is followed by inhibition of intracellular processes, such as protein

synthesis [45], DNA replication [46], or by targeting mitochondria and causing efflux of ATP

[5]. Support for these mechanisms of action comes from investigations of BUF [46], histatins

[47], the P-rich antimicrobial peptide indolicidin [48], and others [4].

Besides these modes of antimicrobial activity, lipid segregation induced by CAPs is

another alternative model which leads to the formation of domains in bacterial membranes.

This model was proposed to explain the membrane permeabilizing action of cyclic RW-rich

hexapeptides [49] (Fig. 8).

Fig. 8. Schematic description of c-RW-induced demixing in a mixed POPG/POPE bilayer. POPG and POPE headgroups are represented by red and blue balls, respectively. c-RW segregates POPG from POPE

creating defects between the formed domains. Taken from ref. [49].

1.4 Small CAPs

1.4.1 Significance of short CAPs

Over the last few decades, thousands of CAPs of various length, amino acid

composition and conformation have been described [50]. Most of them belong to the class of

Introduction

12

-helical peptides. However, unresolved problems of toxicity against eukaryotic cells, the

limited stability against proteolytic digestion in vivo and high production costs prevent using

such CAPs orally and systemically. There is therefore a requirement for the development of

new classes of CAPs with improved activity profiles [2].

Small CAPs rich in particular amino acid residues, such as R, W, and P have gained

high interest as lead compounds [51−55]. They are usually found as small antimicrobial

motifs of much larger natural compounds [56]. The mechanisms of interaction of small and

conformationally constrained peptides with cellular membranes, as well as the key factors that

provide bacterial specificity for these peptides, are much less well understood [57]. Thus,

among others, small RW-rich peptides with improved toxicity against bacteria are interesting

candidates to study the structural motifs and forces responsible for selectivity and may pave

the way to develop new therapeutics with potent activity against multi-resistant bacteria.

The role of R and W residues for biological and bilayer permeabilizing activities has

been described for several sequences. Examples are: the hexapeptide lactoferricin [58],

indolicidin [59], TP [60], Pac-525 [61], the short bovine bactenecins analogs [54], and

synthetic linear RW-rich hexapeptides indentified from combinatorial library studies [62]. On

the level of the lipid membrane, the W residue has a high propensity to insert into the

membrane and partition near the membrane-water interface [63], while positively charged

R residues, with hydrogen bonding properties, provide the basis for peptide interaction with

the anionic components of bacterial membranes. The electric dipole moment and hydrogen

bonding ability of W with both anionic components of lipid membrane, e.g., phosphate groups

and water molecules in the membrane interface, as well as intramolecular cation- electron

interactions between R and W have been suggested to be responsible for favoured partitioning

and the interfacial location of the peptides [64]. These unique properties make peptides, even

if shortened to three amino acid residues, highly active [52].

Furthermore, cationic and bulky hydrophobic amino acids proved to be the best

mimics of the amphipathic topology of the active-site -strands of LPS-binding proteins [65].

W, as a component in many LPS-binding motifs, also points to a significant role of peptide-

sugar interactions for selective toxicity against Gram-negative bacteria. Studies with

lactoferricin-derived peptides and LPS mutant E. coli strains underlined the importance of an

appropriate location of R and W residues for antimicrobial activity [66]. It has been suggested

that the peptides first interact with the negative charges present in the inner core. LPS-

Introduction

13

disorganization finally results in facilitated approach of W residues to the LA as the preferred

hydrophobic binding site [66].

1.4.2 Particular properties of RW-rich hexapeptides

RW-rich hexapeptides derived by structural modifications of the synthetic hexapeptide

Ac-RW (Fig. 9) [53] create one group of small CAPs. The parent sequence, Ac-RW, was

identified by screening of a synthetic combinatorial library [62]. The peptide adopts an

amphipathic structure in a membrane-mimetic environment [67].

Fig. 9. Chemical structures of (A) Ac-RW, and (B) c-RW peptides.

Recently, it has been shown that head-to-tail cyclization of Ac-RW (Fig. 9) distinctly

enhances the peptide’s activity against Gram-negative E. coli [53]. However, in contrast to its

high antimicrobial activity, the cyclic peptide only weakly permeabilizes lipid bilayers

[53,68]. Single amino acid substitutions or replacement of L-amino acid residues by D-

enantiomers were demonstrated to enhance or abolish the antimicrobial activity [53,68,69].

The peptides are able to permeabilize the outer and inner membranes of E. coli [70], but,

interestingly, their activity decreases against wall-deficient L-forms of E. coli. Moreover,

studies with mutant E. coli strains demonstrated that the activity of sequences with three

adjacent aromatic residues, for instance, c-WFW, is reduced upon removal of the O-antigen

Introduction

14

and shortening of the core region of the outer-membrane LPS [70]. This points to a particular

role of LPS in peptide activity and selectivity against E. coli.

Using solution NMR spectroscopy, it was shown that the c-RW consists of two -turn

motifs [71]. On interaction with detergents and lipids, c-RW develops pronounced

amphipathicity compared to its rather flexible structure in water [73]. Molecular dynamic

simulations led to the suggestion that the peptide backbone lies parallel to the lipid bilayer

surface, the positively charged R residues interact with the phosphate groups of the lipids, and

insertion of the aromatic residues into the acyl chain region reduces the permeability barrier

of a DPPC bilayer to water [74]. Furthermore, the peptide induces lipid demixing and

formation of peptide-rich domains in DPPG/DPPE bilayers (Fig. 8) [49].

The observation that many cyclic hexapeptides are similar in conformation, but differ

in the sequence [73] and their antimicrobial activities raises the question of whether specific

amino acid residues are responsible for the cyclization-induced pronounced activity and

selectivity of the RW-rich hexapeptides against Gram-negative bacteria. Studies using

trimesic acid as a template mimicking the peptide backbone have demonstrated an essential

role of the guanidino moiety for bacterial selectivity [74]. However, it seems that not only

charge-driven peptide accumulation at negatively charged E. coli membranes, but also the

exact nature of the interaction of W residues with LPS domains is important to be understood.

Only a few studies have investigated the influence of the aromatic W residues on the bacterial

selectivity of the peptides. So far, c-RW analogs with a lipophilic Nal or a bulky non-aromatic

amino acid residue, i.e., bicyclo[1.1.1]pentane, instead of W were demonstrated to increase

the bactericidal activity of the peptides, whereas introduction of Y or F residues abolished the

activity [53,68,69].

1.5 Surface-tethered peptides

1.5.1 Inhibition of biofilm formation

Biofilm is community of microorganisms growing on a surface and are usually

encased in an extracellular polysaccharide matrix that they themselves secrete [75]. It can

develop on any surface that is exposed to sufficient moisture, such as medical devices,

surgery equipment, implants, food packaging and purification systems (Fig. 10). The

formation of a pathogenic biofilm starts with the adhesion of bacteria or fungi to surfaces by

Introduction

15

non-specific long- and short-range forces [77]. The most common invading pathogens that

cause implant-associated infections include Gram-negative E. coli and Pseudomonas species,

Gram-positive Staphylococci and Candida species [78]. Biofilm is rarely resolved by host

defense mechanisms [75]. In addition, antibiotic therapy typically reverses the symptoms

caused by planktonic cells released from the biofilm, but fails to destroy the biofilm itself

[79]. The factors considered to be responsible for biofilm resistance include: inability of

antimicrobial penetration in all areas of the biofilm, reduced growth rate in biofilm, and

possible expression of resistance genes [80]. Due to their intrinsic resistance to conventional

antibiotic therapy, biofilm formation leads to a significant increase in costs for removing

them. Industrial companies spend billions of dollars a year to control them. To combat this

threat, research must focus on antimicrobial coatings in order to reduce the initiation of

microorganism aggregation on such materials and thus prevent biofilm formation.

Various approaches have been designed in ordrer to develop such biomedical surfaces

including: (i) surfaces covered with bacteria-repellent or anti-adhesive agents using highly

hydrated and close-packed, chain-like molecules, such as PEG [81] or bearing negative

charges [82]; (ii) polymer matrices with incorporated antibiotics (non-covalently), which are

released into the surrounding medium in a controlled manner [83,84]: and (iii) antimicrobial

polymers, which are either prepared by polymerization of constitutive monomers with

therapeutic moieties [85−87] or covalently bound to antimicrobial agents [88−93] (Fig. 11).

However, these strategies suffer from several disadvantages, such as limited affinity of

biomaterials for antibiotics, modification of the mechanical properties of the materials, and a

limited spectrum of therapeutics and active monomers with polymerization-compatible

chemistry. The major obstacle is their hemolytic activity [90,94,95], particularly the toxicity

of surfaces modified with quaternary ammonium, pyridinium and related compounds

[88,96,97] to human cells [98].

The production of surfaces covalently covered with antibiotics is another important

approach to overcome the problem of biofilm formation. Examples of these surfaces are

ampicillin and penicillin attached to expanded poly(tetrafluoroethylene) [99,100].

Introduction

16

Fig. 10. Candida albicans biofilm development on inert surfaces. (1) Inert surface coated with a conditioning film (blue) consisting largely of host proteins. (2) Early attachment and colonization by C. albicans yeast-phase cells (orange). (3) Microcolony yeast basal layer formation, involving stacking of yeasts in the formation of the

microcolonies. (4) Expansion of the biofilm architecture through the development of a hyphal/pseudohyphal layer (turquoise) that protrudes from the inner yeast layer to the outer reaches of the biofilm. Hyphal layer

development occurs simultaneously with the development of the thick layer of matrix material that has engulfed both the hyphal and yeast biofilm layers. Taken from ref. [76].

Fig. 11. Different types of antimicrobial coatings. A) Surfaces covered with ultrahydrophilic molecules, e.g., PEG; B) anionic antimicrobial surfaces, e.g., PAA; C) ultrahydrophobic surfaces, such as lotus-effect surfaces; D) antibacterial coating release, e.g., Ag+, triclosan, Cl2; E) contact killing non-leaching antimicrobial surfaces,

e.g., PVP, TiO2.

Introduction

17

Fig. 12. Oriented grafting of MG 1-Cys derivative on poly(MOE2MA-co-HOEGMA) brushes via a PMPI heterolinker. Taken from ref. [102].

However, the growing emergence of bacterial resistance towards classical antibiotics

is a major drawback to their applicability for the production of antibiotic surfaces. Because of

the bacterial membrane-permeabilizing activities, as well as the low hemolytic activities of

surface-tethered peptides, CAPs have gained high interest as a potential strategy to tackle the

formation of biofilm. Recent examples are: magainin derivatives immobilized on resin beads

[101]; poly(MEO2MA-co-HOEGMA) brushes [102] (Fig. 12) and gold surfaces [103];

immobilized cecropin-MEL hybrid peptide on a variety of different types of substrates such as

amidated polymer brushes, hydrogel and beads [104]; nisin-tethered on block copolymers

made of ethylene oxide and propylene oxide monomers [105]; cellulose membranes covered

with short bactenecins analogs (Bac2A) [106]; a synthetic -helical CAP, namely E14LKK

immobilized with poly(ethylene) [107]; resin beads covered with a -sheet CAP [108]; silica-

and titania-cationic decapeptide (KSL) nanoparticles [109]; titanium surfaces coated with

vancomycin [110], human host defense peptide LL-37 [111] or antimicrobial peptoids [112];

and TP derivatized amphiphilic block copolymers [113]. Inhibition of a biofilm culture of

Pseudomonas aeruginosa, or other oral pathogens, by the human host defense peptide LL-37

or a peptide mimictic based on the structure of MG 2 are examples of this application

[114,115]. The enzymatic degradation of a tethered peptide is significantly slower than the

corresponding soluble peptide [101] and thus the antimicrobial activity mainly originates from

immobilized, as opposed to leached peptides [103,104]. Moreover, the activity of these

Introduction

18

antimicrobial coatings may remain conserved even after heating up to 200 C and over a

broad pH range [116].

1.5.2 Peptide-based biofilm

Recent strategies for the immobilization of CAPs on solid surfaces used either: (i)

covalent linkage via SPPS [101,106,108,116], thioalkylation [102,104,111], EDC/NHS

activation on COOH-enriched surfaces [103,107], disulfide exchange reaction on polymers

using thiolated-peptides [105], or (ii) non-covalent methods using highly specific interactions,

such as the biotin-streptavidin system [106]. CAPs may be tethered randomly on the solid

surfaces or specifically at the C terminus, N terminus or side-chains of the peptides via the -

amino group of selected K residues present within the peptide sequences. However, due to the

random immobilization process there is no control over the orientation of the tethered

peptides.

The pioneer studies by Haynie et al. demonstrated the relationship between peptide

structure and activity of soluble and resin-tethered peptides [101]. It was shown that C-

terminal immobilization distinctly reduced the activity of potent antimicrobial sequences and

that the relationship between the antimicrobial activity and structural properties, such as

amphipathicity, was retained. Furthermore, the structure-related activity profile of the

investigated peptides did not change with immobilization [101]. Studies with a -sheet

antimicrobial peptide attached to PEG-modified beads or directly to the hydrophobic MBHA

resin demonstrated that peptide immobilization via a long spacer stays bactericidal even after

extensive washing [108]. These reports let to the expectation that the activity of immobilized

peptides strongly depends upon the length and kind of spacer between the active sequences

and the solid matrices. However, the flexibility of the peptides, the peptide density on the

surface and the position of immobilization will also influence activity.

The biocidal activity of immobilized peptides may be position-dependent. This means

that the tethered peptides should be tethered on surfaces at a position, which allows effective

interaction between the critical domains of the CAP and the bacterial cell membrane. Support

comes from tethered MG 1 [103], a cecropin-MEL hybrid peptide [104], and an -helical

peptide E14LKK [107]. Randomly immobilized MG 1 on gold surfaces [103], as well as the

peptide C terminally tethered on MEO2MA-co-HOEGMA brushes, showed good activity

against Gram-positive bacteria [102]: the tethered peptide reduced surface adhesion of

Introduction

19

bacteria by more than 50%. In contrast, the antimicrobial activities of a tethered cecropin-

MEL hybrid peptide [104] and an -helical peptide E14LKK [107] are orientation-dependent

with randomly immobilized peptides being almost inactive. The highest specific biological

activities were achieved with the cecropin-MEL hybrid peptide and E14LKK when they were

tethered at the C terminus and N terminus, respectively [104,107]. The screening of

antimicrobial activities of a library of tethered peptides derived from Bac2A analogs against

P. aeruginosa demonstrated that the antimicrobial activity of the immobilized peptides is

influenced by the positioning of hydrophobic and cationic amino acid residues within a

sequence in respect to the site of linkage to the solid surface [106]. For optimal activity, the

hydrophobic and cationic amino acids must occupy places far away and very close to the

linkage site, respectively.

Systematic studies with respect to the influence of the position of peptide coupling, the

distance between the active compounds and the solid surface, as well as peptide density in

relation to an optimized activity, have not been conducted so far. However, because of the

different mode of action of antimicrobial peptides such studies are essential for the selection

of peptides suitable for the generation of antibiotic surfaces.

Moreover, chemical immobilization renders CAPs less flexible and the range of

peptide penetration into the bacterial cell wall is reduced compared to soluble peptides. Due to

the limited ability of tethered peptides to penetrate the bacterial cytoplasmic membrane, it

may allow us to elucidate the membrane selectivity of the peptides. A comparison of activities

of soluble CAPs and surface-tethered compounds on both biological and model membranes

level is expected to provide information about the mode of membrane interaction and

permeabilization. Previous studies with colloidal gold particles coated with gentamicin-BSA

conjugates demonstrated that the covalent immobilization of the aminoglycoside antibiotic

could be used as a strategy to investigate the sites of antibiotic action [117].

Aims of the study

20

2 Aims of the study

2.1 Structural basis of anti-E. coli activity of cyclic RW-

rich hexapeptides

The activity of cyclic RW-rich hexapeptides against Gram-negative E. coli is

determined by the peptide sequence and modulated by LPS in the outer cell wall. In spite of

the amount of experimental data, important issues concerning the particular physicochemical

aspects of W residues within the RW-rich cycles for the interactions with E. coli membranes

are not yet understood. The aim of the first part of the study was to answer the following

questions:

1. Which structural motifs of the cyclic RW-rich peptides are important for

recognition of and interaction with bacterial and model membranes?

2. Are there selective interactions between the aromatic side chain of W residues and

distinct regions of the Gram-negative bacterial outer membrane LPS, which could explain the

high cyclization-induced anti-E. coli activity?

To answer the questions, a set of cyclic hexapeptide analogs of c-WFW carrying

substitutions of W by unnatural aromatic amino acids were synthesized manually. The

substituents differ in terms of hydrophobicity, dipole moment and quadrupole moment

(aromaticity), ability to form hydrogen bonds, and amphipathicity. They include Dht, Igl,

5MeoW, 5fW, 5MeW, 1MeW, and Bal (Fig. 13). In addition, the -amino acid b3-hW was

included to increase the size of the backbone cycle. The activities of the peptides were

evaluated against Gram-negative E. coli (strain DH 5) and Gram-positive B. subtilis (strain

DSM 347) bacteria as well as RBCs. To correlate the biological activity profile with the

driving forces of peptide-membrane interactions, peptides binding to different membrane

model systems were studied by way of ITC. Binding parameters were derived by applying a

surface partition equilibrium model combined with the Gouy–Chapman theory to account for

electrostatic effects at the membrane surface. The phospholipid bilayers used in this study

were composed of POPC or POPC/POPG (3/1 [mol/mol]) to model the electrostatic

properties of erythrocyte and bacterial target membranes, respectively. Furthermore, POPC

bilayers doped with LA, r-LPS, or s-LPS (Fig. 16) were used to assess the contribution of

different regions of outer-membrane LPS to the peptide activity against E. coli.

Aims of the study

21

2.2 Preparation and perspectives of surface-tethered

peptides

It is expected that the antimicrobial activity of tethered peptides is influenced by both

the peptide sequence and the solid surface. The aim of second part of the study was to know:

1. How does immobilization influence the biological and membrane permeabilizing

activities of membrane active peptides?

1.1. How do the physical parameters of the solid material, such as: (i) the spacer

length between the solid surface and the active sequences, (ii) the capacity of the functional

groups on the surface, and (iii) the surface area of solid matrix affect the activities of the

immobilized peptides?

1.2. How does immobilization at different positions of the peptide, C terminus,

N terminus, and side chains, influence the activity spectrum?

2. What will be the effect of immobilization upon the mode of peptide action?

2.1. Could peptide tethering be used as a strategy to get insight into the mode of

action of CAPs?

For this task, CAPs of different sources, structures, and modes of action were tethered

on PEGylated resin beads as a model of solid surfaces. The synthesis resins included:

TentaGel S NH2, TentaGel M NH2, TentaGel MB NH2, HypoGel 400 NH2, and

HypoGel 200 NH2 with various size, capacity and PEG spacer length (Table 9). The

immobilized peptides included: (i) -helical membrane-active peptides, such as a synthetic

model (KLAL) peptide and MK5E (a peptide derived from natural MG 2), which act

according to the carpet-like and toroidal pore mode respectively [44,118]; (ii) MEL, a

membrane-active peptide with the ability to form discrete ion channels [8]; (iii) BUF, a highly

cationic peptide without membrane-permeabilizing activity, which targets intracellular

moieties [46]; and (iv) TP, an RW-rich peptide with a typical turn-turn structure which has

ambiguous modes of action ranging from membrane lysis to interaction with intracellular

targets [119,120]. Standard SPPS, thioalkylation, and oxime-forming ligation strategies were

used to immobilize the peptides at the C and N termini and via different side-chain positions.

In order to demonstrate the suitability of the peptides for preparation of bioactive surfaces,

Aims of the study

22

antimicrobial and hemolytic activities of tethered peptides were evaluated against E. coli and

B. subtilis, as well as RBCs, and compared to that of soluble peptides. Model membranes such

as POPC, POPC/POPG (3/1 [mol/mol]), POPC/POPG (1/3 [mol/mol]), loaded with calcein

were prepared to assess the lipid bilayer-permeabilizing activities of soluble and tethered

peptides. These results were correlated to their biological activities in order to study the

influence of tethering upon the peptides mode of action.

Results and discussion

23

3 Results and discussion

3.1 W-substituted c-WFW analogs

In order to investigate the influence of the physicochemical properties of W residues

upon the bacterial selectivity of c-WFW and to analyze the interaction with lipid bilayers

mimicking the inner and outer membranes of bacteria, W residues were replaced in the

sequence with unnatural amino acids, such as Dht, Igl, 1MeW, Bal, 5MeW, 5MeoW, 5fW and

b3-hW (Fig. 13). Each modification endows the cyclic peptide with a unique property

(Table 2). The biological activities of the peptides were correlated with their affinity for lipid

bilayers doped with LPS mutants in order to indentify structural motifs of the peptides and

interaction partners on cellular level, which are responsible for the recognition of E. coli.

3.1.1 Description and physicochemical properties of W-analogs

The aromatic side chain W is regarded as a highly hydrophobic residue based on the

Liu-Deber hydrophobicity scale [121], characterized by a dipole moment of ~ 2.1 D in

magnitude, which is directed from N-1 in the five-membered ring to C-5 in the six-membered

ring of indole [122] and able to form hydrogen bonds.

Fig. 13. A) Schematic structure of cyclic peptide with the positions for the desired modifications, and (B) chemical structure of the amino acids analogs of W used in this study.

Dht is the product of the reduction of W, is no longer planar in character and

introduces steric, as well as charge distribution changes. With a changed distribution of


24

electrons, it has neither the aromaticity nor the dipole moment of W, but hydrogen bonding

ability is conserved. As the aromaticity of W has been proposed as the reason for its water-

lipid interfacial preference [63], Dht does not seem to position at this region. Igl also bears a

fused-ring aromatic side chain, which is; however, closer to the backbone compared to Dht

and incapable of hydrogen bonding. Similar to Dht, Igl is not expected to lie at the lipid-water

interfacial region; however, it is more hydrophobic than Dht (Fig. 14, Table 3). The hydrogen

bonding ability of W is blocked in the case of 1MeW by the more hydrophobic and bulky

methyl group. However, 1MeW has the amphipaticity of W and a dipole moment (~ 2.2 D)

with similar direction and magnitude [122]. Bal has the same size as the indole ring, but

because of the lower electronegativity of the sulfur atom in the five-membered ring, its dipole

moment is reduced compared to W (Table 2). It has no considerable amphipathic structure

and does not have the hydrogen bonding characteristics of W [123]. Both 1MeW and Bal

residues are more hydrophobic than W. This appeared in the higher tR-values of c-

(1MeW)F(1MeW) and c-(Bal)F(Bal) compared to c-WFW (Fig. 14, Tables 3).

Table 2. Changes in the physicochemical characteristics of W analogs.

Physicochemical characteristics of W-analogs*

Degree of change Hydrogen bonding Dipole moment Quadrupole moment Amphipathicity

5MeoW 5fW 5MeoW 5MeoW

> W 5fW

Dht b3-hW b3-hW b3-hW 5fW 5MeoW

= W 5MeW 5MeW 5MeW b3-hW 5MeW

1MeW 1MeW 1MeW Dht

Bal

Igl Dht Dht Igl

< W 1MeW Igl Igl Bal

Bal Bal 5fW *The classification is based on the chemical structures of the residues. Their influence on peptide hydrophobicity and amphipathicity as reflected in the RP-HPLC retention behavior is shown in Table 3.


25

Unlike 1Metrp, 5Metrp is potentially able to form hydrogen bonding with water or

anionic phosphate and carboxylate groups of lipid membranes. However, the quadrupole

moment (aromaticity) and the dipole moment of 5MeW are similar to that of W and 1MeW in

magnitude and direction [122,124]. In contrast, the methoxy group and fluorine atom with

their electron-withdrawing effect at C-5 will enhance the dipole moment of indole. However,

the density of negative charges above and below the plane of the indole ring is weaker in the

case of 5fW, which will lead to a lower quadrupole moment compared to W. On the other

hand, the methoxy group will push electrons into the indole ring plane resulting in

strengthening the -electron system and quadrupole moment compared to W [124].

Substitution of the hydrogen at C-5 by the methoxy group and fluorine atom will also enhance

the hydrogen bonding character of the NH moiety. In addition, the methoxy group and

fluorine atom can form hydrogen bonds with water molecules at the lipid bilayer interface.

Incorporation of b3-hW with the same side chain as W enhances the size and likely the

flexibility of the backbone ring (Fig. 13). Therefore, the lipid-bound peptide might adopt

conformations different from the parent cyclic peptide (c-WFW) with a different degree of

amphipathicity.

3.1.2 Cyclic peptide synthesis and their HPLC characterization

The sequences synthesized for this study (Table 3) are based on the previously

described hexapeptide c-WFW with three adjacent aromatic and charged residues [53,68].

Key features, such as the number and distribution of the three R and non-charged residues

were maintained.

The hydrophobicity/amphipathcity of c-WFW is characterized by tR = 18.83 min in

RP-HPLC (Fig. 14, Table 3). Because of the protonation of NH at physiological pH, the

peptide with two Dht residues is much more hydrophilic than c-WFW. Also, c-(Igl)F(Igl) is

highly hydrophilic and characterized by tR = 15.91 min in RP-HPLC. c-(b3-hW)F(b3-hW) is

slightly more hydrophobic than c-WFW as the result of two extra methylene groups. The Bal-

containing peptide is the most hydrophobic one in this series. Introduction of 5MeoW, 5fW,

1Metrp, and 5MeW only slightly enhances the hydrophocitiy compared to the parent peptide

(19.08 min < tR < 20.41 min).


26

Table 3. Amino acid sequences, calculated and observed molecular masses, and tR in RP-HPLC of c-WFW, W-substituted analogs, and the linear Ac-WFW.

Molecular mass (Da.)

Peptide denotation* Amino acid sequence calculated observed tR (min)

c-(Dht)F(Dht)† cyclo-RRR(Dht)F(Dht) 992.2 992.5 9.92

c-(Igl)F(Igl) cyclo-RRR(Igl)F(Igl) 961.8 962.5 15.91

c-WFW cyclo-RRRWFW 988.2 988.5 18.83

c-(5MeoW)F(5MeoW) cyclo-RRR(5MeoW)F(5MeoW) 1048.2 1049.1 19.08

c-(5fW)F(5fW) cyclo-RRR(5fW)F(5fW) 1024.2 1025.3 19.92

c-(b3-hW)F(b3-hW) cyclo-RRR(b3-hW)F(b3-hW) 1016.2 1016.6 20.06

c-(5MeW)F(5MeW)† cyclo-RRR(5MeW)F(5MeW) 1016.2 1016.6 20.37

c-(1MeW)F(1MeW) cyclo-RRR(1MeW)F(1MeW) 1016.2 1016.8 20.41

c-(Bal)F(Bal) cyclo-RRR(Bal)F(Bal) 1022.2 1022.4 22.03

Ac-WFW Ac-RRWFWR-NH2 1047.2 1047.5 16.58 *The purities of the products were more than 95% (Fig. 14).

†These unnatural amino acids are not enantiomerically pure. See section for Abbreviations.

3.1.3 Characterization of cyclic peptides by CD

To get structural information on representative cyclic peptides in comparison to the

linear Ac-WFW, CD spectra of the peptides containing N-1 and C-5 substituted W analogs, c-

(b3-hW)F(b3-hW), and c-(Bal)F(Bal), as well as the parent peptide, were recorded in

phosphate buffer, a mixture of buffer and TFE and in the SDS micelle- and POPG liposome-

bound state (Fig. 15). While studies in buffer provide information on the structural flexibility

of peptides in aqueous solution, TFE represents a solvent which induces intramoleular and

intermolecular hydrogen bonding and thus favours helix formation of linear peptides [125].

TFE solvent conditions were taken to monitor the ability / propensity of peptides to adapt a

secondary structure [17]. The anisotropic nature of SDS micelles and POPG bilayers represent

more suitable models of cell membranes and the high negative charge provides the basis for

high peptide binding [126,127].

The spectra of c-WFW dissolved in buffer shows a negative band and a shoulder at

200 and 220 nm, respectively (Fig. 15). The ellipticities at 200 nm and below originate from

the peptide bonds and thus changes in this spectral region reflect the properties of the

backbone. Less negative and positive ellipticities below 200 nm are associated with


27

constrained structures. In the wavelength region of 220−230 nm, contribitions of the peptide

bond and the aromatic side chains superimpose. In the presence of TFE only minor spectral

changes were observed for c-WFW, thus confirming a rather limited conformational

flexibility of the cycle. Furthermore, interaction with SDS and POPG had minor influences

upon the conformation.

Fig.14. HPLC profile of c-WFW, W-substituted analogs, and the linear Ac-WFW. Except for the linear sequence, the panels were organized according to the increase in the tR-values of the cyclic peptides. The

chromatogram for c-(5MeW)F(5MeW) clearly displays mixtures of diastereomers.


28

Fig. 15. Far-UV CD spectra of cylic peptides in phosphate buffer (green), 1:1 TFE/buffer [v/v] (red),

SDS bound (black), and POPG SUVs lipid bound (yellow) (T = 20 C).

Ac-WFW reveals a positive band at 225 nm and decreasing ellipticity values down to

200 nm and below, which are characteristic of small random-coil peptides. In the presence of

TFE, Ac-WFW showed spectral properties comparable to c-WFW though with different band

intensities. Binding to POPG vesicles shifts the negative band from 220 to ~ 225 nm. The

changes in the spectral characteristics of Ac-RW are comparable to those observed for c-RW

which have been attributed to restrictions in the backbone structure and changes in the

environment of the aromatic residues [53]. The increase in band intensity in the 200 nm

region observed for SDS-bound c-WFW, in comparison to the solution structure, could also

be associated with a reduction of flexibility in the cyclic backbone. The derived NMR

structure of c-RW and Ac RW in micelle- and lipid-bound states [67,71] indicates that the

amphipathic structures of the cyclic peptides were little modified with sequence modifications

[73]. Thus, the CD spectra of c-WFW is assumed to reflect the structures derived from NMR

measurements: a hydrophobic cluster of the aromatic residues in contact to the hydrophobic


29

chains of SDS and POPG and the backbone and cationic side chains exposed to the negative

charges of SDS and POPG. Introduction of 5MeoW, 5fW, 5MeW and 1MeW had little

influence upon the spectral characteristics of the cycles. However, following introduction of

Bal, the contribution of the aromatic side chain to the spectrum was lost. The pronounced

band intensity of the SDS-bound peptide at lower wavelengths (210 nm), where the

contribution of the backbone amid bonds predominates suggests that binding also reduces the

number of backbone conformers of c-(Bal)F(Bal).

Introduction of b3-hW enlarges the ring size and is expected to enhance the backbone

flexibility of the hexapeptide. The b3-hW-containing peptide shows a sharp negative band at

~ 225−230 nm, both in aqueous environment and when bound to POPG membranes (Fig. 15).

This pronounced conformational change might be due to the disturption of the -turn motifs

in c-WFW [73] and induction of an amphipathic conformation different from that of c-WFW.

Interestingly, in the SDS-bound state, spectral characeristics comparable to the other cyclic

peptides, with a pronounced band in the 200 nm region, was observed for c-(b3-hW)F(b3-

hW). This suggests that this peptide is also able to assume an amphipathic structure as

suggested for the cyclic parent sequence and other analogs. Recent studies with analogs of

Gramicidin S, with ring sizes ranging from 10 to 16 amino acid residues, confirmed the

importance of amphipathicity induced by the -turn/-sheet structure in interaction with

membranes [128]. Disruption of the structure by increasing the size of the cyclic peptide

weakened the peptides’ interactions with POPC vesicles and reduced their hemolytic,

antimicrobial, and antifungal activities.

3.1.4 Antibacterial and hemolytic activities

The antimicrobial activities of c-WFW, its analogs and the linear sequence (Ac-WFW)

are summarized in Table 4. In general, the activity of the cyclic peptides was higher against

B. subtilis than E. coli and correlated with the peptide hydrophobicity.

While the flexible Ac-WFW and hydrophilic peptides, i.e, c-(Dht)F(Dht) and c-

(Igl)F(Igl), showed MICs against B. subtilis in the range of 2550 M, the cyclic peptides,

with tR-values ranging between 18.93 min and 22.03 min, were highly active

(1.6 M < MIC < 6.3 M). The reduction in activity of c-(b3-hW)F(b3-hW) by one dilution

step compared to c-WFW correlates with an enhanced flexibility in the cyclic backbone.


30

Table 4. Antimicrobial activities, and hemolytic activities of the peptides used in this study.

MIC (M)*

Peptide denotation B. subtilis (DSM 347) E. coli (DH 5) Hemolysis for RBCs†

c-(Dht)F(Dht) 50 200 1

c-(Igl)F(Igl) 25 200 3

c-WFW 3.1 3.1 6

c-(5MeoW)F(5MeoW) 3.1 12.5 1

c-(5fW)F(5fW) 1.6 3.1 ND‡

c-(b3-hW)F(b3-hW) 6.3 50 1

c-(5MeW)F(5MeW) 3.1 6.3 5

c-(1MeW)F(1MeW) 3.1 6.3 27

c-(Bal)F(Bal) 1.6 12.5 70

Ac-WFW 50 400 ND‡ *Values represent the means of three independent experiments performed in triplicate. Standard deviations after 17 h of cell incubation at 37 C were < 5% (Fig. 39).

†Values represent the percentage release of hemoglobin from human RBCs upon incubation with cyclic peptides at cP = 200 M. Hemolytic activity was monitored by measuring the absorbance at = 540 nm.

‡ND, not determined.

The activity spectrum of the peptides against E. coli was more complex. The activity

of Ac-WFW, which is very low (MIC = 400 M), increased > 130-fold after cyclization. The

MIC of c-WFW against E. coli was 3.1 M and the same for B. subtilis. Increasing the ring

size of the cyclic peptide in c-(b3-hW)F(b3-hW) caused 16-fold reduction in anti-E. coli

activity (MIC = 50 M). The least hydrophobic peptides, c-(Dht)F(Dht) and c-(Igl)F(Igl),

showed little activity against E. coli (MIC = 200 M), which supports a significant role of

hydrophobicity and the amphipathic nature of W residues upon antimicrobial effect.

Interestingly, c-(5MeoW)F(5MeoW) and the most hydrophobic peptides, i.e., c-(Bal)F(Bal),

were also much less active against the Gram-negative strain than c-WFW, an observation

different to B. subtilis where the MIC values were reduced or conserved, respectively. The

observation for the antimicrobial activities of c-(Bal)F(Bal) is in contrast to the results on the

bactericidal activity of a 15-residue Bal-modified lactoferricin derivative, which exhibited

higher activity than the parent peptide against S. aureus and E. coli [123]. c-(5fW)F(5fW) had


31

the same activity as c-WFW whereas c-(5MeW)F(5MeW) and c-(1MeW)F(1MeW), two

peptides with comparable high hydrophobicity, were less active against E. coli.

Except for the most hydrophobic peptides, c-(Bal)F(Bal) and c-(1MeW)F(1-MeW),

causing 70% and 30% hemoglobin release from human RBCs repectively at 200 M, all

peptides showed no significant hemolytic activity (Table 4). The different susceptibilities of

cells can be explained on the basis of peptide accumulation at the lipid matrices of the target

membranes driven by electrostatic interactions.

3.1.5 Cyclic peptide binding to lipid bilayers determined by ITC

3.1.5.1 Lipid bilayers as model of biological membranes

The envelope of Gram-negative E. coli bacteria consists of an inner (cytoplasmic)

membrane with a composition of 80% POPE, 15% POPG, and 5% cardiolipin [23], and a

highly asymmetric outer membrane with LPS ( 90%) in the outer leaflet and POPE as the

main components of the inner leaflet. To study peptide binding to lipid membranes, POPC,

POPC/POPG (3/1 [mol/mol]), POPC/LA (12/1 [mol/mol]), and POPC mixed with two E. coli

LPS chemotypes (POPC/Rd-LPS and POPC/s-LPS ratio 12/1) (Fig. 16), were chosen to

mimic the charge properties of mammalian and Gram-negative cellular inner (cytoplasmic)

and outer membranes [127].

Fig. 16. Schematic structure of different LPS chemotypes from E. coli.

Due to POPE having a negative curvature strain, which might interfere in liposome

formation, especially in the presence of natural lipids, such as LA and LPS [21], POPC was

used as the zwitterionic phospholipid for the preparation of mixed vesicles. LA is the


32

conserved part of LPS and has two divalent phosphate anions ([─OPO3]-2), and thus the ratio

of negative charges of phosphate anions in POPC/LA (12/1 [mol/mol]) vesicles is the same as

POPC/POPG (3/1 [mol/mol]). The main difference between the two LPS chemotypes comes

from the composition of the polysaccharide part. The negatively charge mono ([─OPO3─]−)

and divalent phosphate anions and carboxylate groups ([─CO2]−), attached at various

positions to heptose moieties, are located at the inner core oligosaccharide part of LPS,

resulting in the same number of negative charges for r-LPS and s-LPS. s-LPS has a long

polysaccharide region of repeating oligosaccharide units (O-antigen region) attached to the

core polysaccharide. In contrast, r-LPS belongs to deep rough mutants with the sugar moieties

limited to the inner core oligosaccharide [28]. In this study, only the total negative charges of

phosphates anions (nine charges) were considered for binding studies.

3.1.5.2 Peptide accumulation as reflected by apparent binding

Influence of lipid composition upon binding

Figs. 17 and 18 display typical ITC titration traces of small aliquots of POPC and

POPC/POPG (3/1 [mol/mol]) SUV suspensions into the calorimeter cell containing the

peptides at 37 C. The surface underneath each signal represents the heat flow after an

individual titration step. In general, the heat of binding of the cyclic peptides decreases with

the number of injections because less and less peptide is available for binding. The signals

finally approach the heat of dilution indicating that virtually all the peptide is bound to lipid

vesicles. Comparable traces, but of enhanced signal intensity, were observed for peptide

titration with POPC/POPG SUVs showing a much enhanced interaction of the cationic

peptides with the negatively charged lipid system due to strong electrostatic interactions

(Figs. 17, 18).

To investigate the role of LPS in peptide selectivity against E. coli, peptide binding to

POPC bilayers containing LA, r-LPS or s-LPS at a molar ratio of 12/1 was studied. The ITC

traces for peptide interaction with POPC/s-LPS (12/1 [mol/mol]) are shown as an example in

Fig. 19. Fig. 20 demonstrates the binding isotherms for the lipid systems derived from the ITC

traces, which gives Rb as function of cP,f. Except for the most hydrophilic peptide, c-

(Dht)F(Dht), the binding curves for peptide interaction with the POPC containing LA, r-LPS,

and mixed POPC/POPG bilayers were almost comparable (Fig. 20).


33

r-LPS contains roughly nine negatively charged phosphate groups and thus peptide

binding to POPC/r-LPS bilayers was expected to be higher than to POPC mixed with LA,

which bears two phosphate groups. However, it was found that the increase in negative charge

in r-LPS-doped bilayers compared to the POPC/LA system only slightly enhanced Rb

(Fig. 20). In contrast, in the presence of s-LPS, peptide binding distinctly increased and the Rb

values were doubled compared to r-LPS and LA with reduced size of the carbohydrate moiety

(Fig. 20). This binding behavior correlates with reduced antimicrobial activity against O-

antigen- and outer core-deficient LPS mutant E. coli strains, which was observed for RW-rich

cyclic hexapeptides with three adjacent aromatic residues [70].

Fig. 17. ITC traces (differential heating power vs. time) of the titration of Ac-WFW and cyclic peptides with POPC SUVs (T = 37 C). Each titration step corresponds to the injection of

10 L of 40 mM lipid suspension in phosphate into 40 M

peptide solution (except the first injection, which was only 5 L).


34

Role of sequence composition upon binding

Low binding of the linear and the two most hydrophilic cyclic peptides to POPC is

reflected by Rb values (~ 1 × 10-2 mol/mol at cP,f = 30 M; calculated according to the Eq. 4

shown in the experimental section) (Fig. 20). The Rb values (at cP,f = 30 M) follow the order:

Ac-WFW c-(Igl)F(Igl) c-(Dht)F(Dht) < c-(5MeoW)F(5MeoW) c-(b3-hW)F(b3-hW) <

c-WFW c-(1MeW)F(1MeW) < c(5fW)F(5fW) c-(5MeW)F(5MeW) < c-(Bal)F(Bal)

(Fig. 20). The negative charge in POPC/POPG bilayers enhanced the Rb, but also reduced

differences in Rb between the individual peptides (Fig. 20).

Fig. 18. ITC traces (differential heating power vs. time) of the titration of cyclic peptides with POPC/POPG (3/1 [mol/mol]) SUVs (T = 37 C). Each titration step corresponds to the injection of 6 L of 20 mM lipid suspension into 40 M peptide solution in the phosphate buffer except the first injection being 3 L.


35

With respect to the magnitude and sequence dependency of the thermodynamic

parameters, the binding of the cyclic peptides to POPC/LA was comparable to that of

POPC/POPG lipid system. Interestingly, for POPC containing r-LPS and s-LPS, peptide

sequence-related differences were reduced (Fig. 20). An exception is Ac-WFW; its binding to

liposomes containing LA, r-LPS and s-LPS was as low as to POPC and POPC/POPG SUVs.

These differences might be due to favorable contributions of the sugar moieties of LPS

to peptide binding. In a recent study, the interactions between carbohydrates and aromatic

groups have been described in terms of the hydrophobic effect, dispersion forces and

carbohydrate electron interaction [129]. Carbohydrates interact in a favorable manner with

Fig. 19. ITC traces (differential heating power vs. time) of the titration of Ac-WFW and cyclic peptides with POPC/s-LPS (12/1 [mol/mol]) SUVs (T = 37 C). Each titration step

corresponds to the injection of 3 L of a 5 mM lipid suspension

in phosphate buffer into a 40 M peptide solution.


36

peptides and proteins via stacking involving aromatic side chains [129]. The -electron

distribution, surface area, and flexibility of the aromatic systems affect the magnitude of the

interaction. In recent studies on a -hairpin peptide containing a W residue, as well as a

glucosyl or galactosyl analog of serine, strong intramolecular carbohydrate– electron

interaction (2.13.3 kJ/mol; stronger than – or cation– interactions) were found to

stabilize the peptide’s secondary structure [129].

Fig. 20. Binding isotherms of cyclic and linear peptides for (□) POPC, (○) POPC/POPG (3/1 [mol/mol]),

(▲) POPC/LA (12/1 [mol/mol]), (■) POPC/r-LPS (12/1 [mol/mol]), and (●) POPC/s-LPS (12/1 [mol/mol])

SUVs (T = 37 C). It is assumed that the cationic peptides cannot cross the bilayers and only 60% of the total lipid amount is accessible for binding. The data were calculated by combining a surface partitioning equilibrium

with Gouy–Chapman theory. The fit parameters (K0, ΔH°, and zP) are listed in Tables 5 and 6.


37

3.1.5.3 Peptide partitioning in lipid bilayers

Influence of lipid composition upon binding

Figs. 21 and 22, and Tables 5 and 6 show the G° and K0 values of the individual

peptides for binding to lipid bilayers. The best fits of binding data were obtained for zP

smaller than the nominal charge of peptides. Peptide partitioning (G° and K0 values) into

POPC, POPC/POPG and POPC/LA did not differ. This indicates that the hydrophobic

contribution to binding to these lipid systems is identical and independent upon interactions

between the cationic R residues and the negatively charged lipid phosphate groups. However,

in the presence of sugar moieties of the inner core and O-antigen of LPS, peptide partitionig

increased. As shown in Fig. 22A, the negative values for G° are arranged in the following

order: POPC/LA < POPC/r-LPS << POPC/s-LPS. The K0 values for peptide binding to

POPC/s-LPS bilayer were almost one order of magnitude higher than for binding to bilayers

doped with O-antigen-deficient r-LPS and LA (Table 6). Based upon these observations, it

can be concluded that hydrophobic peptide-LPS interaction is essential for an efficient

transport across the bacterial outer membrane, but that LA does not act as a specific activity

modulating binding site of the hexapeptides. This is in accordance with observations with

lactoferricin peptides suggesting that the tight fatty acid packing in LA is not the primary site

of interaction [130].

Fig. 21. Thermodynamic parameters of binding of Ac-WFW and the cyclic hexapeptides to lipid bilayers

(T = 37 C). (A) G° for binding to (□) POPC and (○) POPC/POPG (3/1 [mol/mol]) SUVs. (B) H° and TS° as ( ) and ( ), respectively.


38

Role of sequence composition upon binding

Binding to POPC followed the order: Ac-WFW c-(Igl)F(Igl) c-(Dht)F(Dht) < c-

(5MeoW)F(5MeoW) c-(b3-hW)F(b3-hW) c-WFW < c-(1MeW)F(1MeW)

c(5fW)F(5fW) c-(5MeW)F(5MeW) c-(Bal)F(Bal) (Table 5). The free energy of binding

of Ac-WFW, c-(Igl)F(Igl) and c-(Dht)F(Dht) is low, while G° values for the more

hydrophobic peptides (tR > 18 min) range between –31 and –35 kJ/mol and are almost

identical. Among these, c-(b3-hW)F(b3-hW) shows lowest partitioning (G° ~ –31 kJ/mol),

indicating the role of conformational constraints of the cyclic peptides in binding. As shown

in Table 5 and Fig. 21A, the contribution of H° and the entropy (–TS°) to lipid bilayer

partitioning is highly variable. Both components of G° are comparable for binding of the

highly hydrophilic c-(Dht)F(Dht) and c-(Igl)F(Igl), and the most hydrophobic c-(Bal)F(Bal) to

POPC. The common feature of Dht, Igl, and Bal residues is a reduced dipole moment

compared to W; however, unlike Igl and Bal, Dht is as amphipathic as W. The small enthalpic

contribution to POPC/POPG bilayer binding for Dht-containing peptides is compensated by a

more favorable entropic term (Table 5), which is in agreement with the classical hydrophobic

effect [131]. This effect was observed for the antimicrobial peptide dicynthaurin as well

[132]. The authors considered water and counterion release from the peptide and a sodium

binding equilibrium at the lipid headgroups as the major driving forces for peptide–membrane

interactions. The binding reaction of the other peptides including the linear Ac-WFW to both

POPC and POPC/POPG systems is driven by enthalpy with H° values varying between

about ~ –40 to ~ –20 kJ/mol (Table 5, Fig. 21). The enthalpy contribution is highest (> –

30 kJ/mol) for c-(5fW)F(5fW) and c-(5MeoW)F(5MeoW) and followed by c-WFW and Ac-

WFW. The pronounced H° of c-(5MeoW)F(5MeoW) and c-(5fW)F(5fW) correlates with a

slightly enhanced peptide hydrophobicity and an enhanced hydrogen bonding tendency in

comparison to the cyclic parent peptide (Tables 3, 5). The high H° contribution is associated

with a low positive and negative value of TS° (Fig. 21B). H° of c-(b3-hW)F(b3-hW), c-

(5MeW)F(5MeW), and c-(1MeW)F(1MeW) binding ranges between ~ –20 and –27 kJ/mol

and the contribution of entropy is about 6−12 kJ/mol. The common feature of these cyclic

peptides is a mean hydrophobicity reflected by a tR of about 20 min (Table 3). Except the Dht,

Igl- and Bal-containing cyclic peptides, peptide binding to POPC and POPC/POPG lipid

bilayers is in good agreement with the nonclassical hydrophobic effect as described for other

CAPs [55,133,134].


39

These results demonstrate that low peptide hydrophobicity and conformational

flexibility of the peptides reduced binding whereas the hydrogen bonding ability of the

W residue and its dipole and quadrupole moments did not make any distinguished

contribution to bilayer binding, as reflected by studies with c-(1MeW)F(1MeW), and c-

(5fW)F(5fW) / c-(5MeoW)F(5MeoW), respectively (Fig. 21A, Tables 3 and 5). Studies with a

1MeW-modified lactoferricin analog revealed enhanced membrane binding and showed

1MeW to be aligned at the membrane interface with an extent of motion similar to that of W

[135]. Furthermore, the influence of dipole and quadrupole moments became apparent in

intramolecular interactions between the -electrons of the indole ring of W with the positively

charged guanidino moiety of R in RW-rich peptides, which were suggested to stabilize the

structure of CAPs and enhance membrane binding [136,137]. Because of the higher

quadrupole moment (aromaticity) of the indole ring in c-(5MeoW)F(5MeoW) compared with

c-(5fW)F(5fW) [124], a higher affinity of the former peptide for lipid bilayers was expected

[136,137]. However, the opposite was observed (Fig. 21A and Table 5). This underlines the

contribution of other driving forces, such as the dominant role of hydrophobicity for insertion

of the RW-rich cyclic hexapeptides into a phospholipid membrane.

Fig. 22. Thermodynamic parameters of the binding of Ac-WFW and the cyclic hexapeptides to lipid bilayers doped with LA, r-LPS, and s-LPS (T = 37 C). (A) G° for binding to (▲) POPC/LA

(12/1 [mol/mol]), (■) POPC/r-LPS (12/1 [mol/mol]), and (●) POPC/s-LPS (12/1 [mol/mol]) SUVs. (B) H° and TS° as ( ) and ( ), respectively.


40

Table 5. Thermodynamic parameters for peptide binding to POPC and POPC/POPG (3/1 [mol/mol]) SUVs (T = 37 C).

Lipid bilayers (SUVs)

Peptide* POPC* POPC/POPG (3/1 [mol/mol])*

H°

(kJ/mol) −TS°

(kJ/mol) G°

(kJ/mol) K0 (M

-1) zP† H°

(kJ/mol) −TS°

(kJ/mol) G°

(kJ/mol) K0 (M

-1) zP†

c-(Dht)F(Dht) −12.1 −14.5 −26.6 5.4 × 102 1.0 −11.4 −19.0 −30.4 2.4 × 103 0.5

c-(Igl)F(Igl) −13.0 −11.9 −24.9 2.8 × 102 1.0 −20.0 −7.8 −27.8 8.9 × 102 1.8

c-WFW −31.2 −1.8 −33.0 6.7 × 103 1.3 −23.5 −11.0 −34.5 1.2 × 104 1.2

c-(5MeoW)F(5MeoW) −40.0 8.2 −31.8 4.1 × 103 1.3 −37.6 4.3 −33.3 7.5 × 103 1.8

c-(5fW)F(5fW) −40.7 6.0 −34.7 1.3 × 104 1.4 −41.6 6.2 −35.4 1.7 × 104 1.8

c-(b3-hW)F(b3-hW) −19.8 −10.7 −30.5 2.5 × 103 1.0 −23.3 −8.2 −31.5 3.7 × 103 1.2

c-(5MeW)F(5MeW) −21.8 −12.2 −34.0 9.9 × 103 1.2 −25.9 −9.0 −34.9 1.4 × 104 1.4

c-(1MeW)F(1MeW) −27.6 −7.2 −34.8 1.3 × 104 1.6 −27.7 −6.1 −33.8 9.0 × 103 1.4

c-(Bal)F(Bal) −18.9 −15.2 −34.1 1.0 × 104 1.0 −16.4 −17.2 −33.6 8.4 × 103 0.7

Ac-WFW −30.0 4.0 −26.0 4.4 × 102 1.9 −28.4 −0.2 −28.6 1.2 × 103 1.7 *The peptide concentration was 40 M, the concentration of POPC and POPC/POPG (3/1 mol/mol) in the injection syringe was 40 mM and 20 mM, respectively.

†The effective charge number of the peptides corresponds to the best fits of the experimental data using the surface partition equilibrium in combination with Gouy–Chapman theory.


41

Table 6. Thermodynamic parameters for peptide binding to POPC/LA (12/1 [mol/mol]), and POPC/r-LPS (12/1 [mol/mol]), and POPC/s-LPS (12/1 [mol/mol]) SUVs (T = 37 C).

Lipid bilayers (SUV)

Peptide* POPC/LA (12/1 [mol/mol])* POPC/r-LPS (12/1 [mol/mol])* POPC/s-LPS (12/1 [mol/mol])*

H°

(kJ/mol)

TS°

(kJ/mol)

G°

(kJ/mol)

K0 (M-1) zP H°

(kJ/mol)

TS°

(kJ/mol)

G°

(kJ/mol)

K0 (M-1) zP H°

(kJ/mol)

TS°

(kJ/mol)

G°

(kJ/mol)

K0 (M-1) zP

c-(Dht)F(Dht) 10.0 19.4 29.4 1.6 × 103 1.0 4.1 30.2 34.3 1.1 × 104 0.1 7.7 28.9 36.6 2.7 × 104 1.0

c-(Igl)F(Igl) 9.30 18.0 27.3 7.3 × 102 1.0 11.2 20.4 31.6 3.8 × 103 0.0 15.0 20.4 35.4 1.7 × 104 1.2

c-WFW 27.1 4.7 31.8 4.2 × 103 1.7 19.5 14.5 34.0 9.9 × 103 1.6 28.7 7.0 35.7 1.9 × 104 0.8

c-(5MeoW)F(5MeoW) 40.4 10.8 29.6 1.8 × 103 2.2 30.0 0.8 30.8 2.8 × 103 1.9 36.0 0.3 35.7 1.9 × 104 1.2

c-(5fW)F(5fW) 33.7 1.1 34.8 1.3 × 104 1.8 26.7 7.0 33.7 8.7 × 103 1.6 28.0 10.6 38.6 5.8 × 104 0.9

c-(b3-hW)F(b3-hW) 18.3 12.2 30.5 2.5 × 103 1.2 17.6 13.8 31.4 3.5 × 103 1.3 20.5 14.3 34.8 1.3 × 104 0.7

c-(5MeW)F(5MeW) 20.2 12.0 32.2 4.8 × 103 1.2 25.0 6.0 31.0 3.0 × 103 1.3 30.0 4.5 34.5 1.2 × 104 0.7

c-(1MeW)F(1MeW) 22.6 11.2 33.8 9.1 × 103 1.8 23.5 9.7 33.2 7.0 × 103 1.6 30.7 5.2 35.9 2.0 × 104 0.9

c-(Bal)F(Bal) 15.4 18.3 33.7 8.7 × 103 1.0 22.9 9.3 32.2 4.8 × 103 1.2 28.4 6.7 35.1 1.5 × 104 0.5

Ac-WFW 29.4 5.3 24.1 2.1 × 102 2.3 30.0 3.0 27.0 6.5 × 102 0.5 33.3 2.3 31.0 3.0 × 103 1.7 *The peptide concentration was 40 M, for that of POPC/LA (12/1 [mol/mol]) and vesicles composed of LPS were 20 mM and 5 mM, respectively.


42

Binding of the cyclic sequences to LA systems was observed to correlate with peptide

hydrophobicity: G° values gradually decreased from ~ –28 to ~ –35 kJ/mol (Fig. 22A).

Interestingly, partitioning of c-(Dht)F(Dht) and c-(Igl)F(Igl) into POPC/LA became

entropically driven (Table 6). The G° values for interaction with r-LPS containing bilayers

scatter around –32 kJ/mol while they seem to increase with increasing tR for POPC/s-LPS

lipid bilayers. Compared to the POPC/LA system, r-LPS doped bilayers favored partitioning

of the most hydrophilic peptides (Dht- and Igl-containing cycles). The interaction of the most

hydrophobic, c-(Bal)F(Bal), with the three lipid systems is least differentiated, as is reflected

by comparable G° and K0 values (Fig. 22A, Table 6). Furthermore, the ring size has little

effect on G°.

Partitioning of hydrophilic c-(Dht)F(Dht) and c-(Igl)F(Igl) into the LPS-doped lipid

vesicles was entropy-driven as reflected by the large positive values for TS°, whereas

binding of other cyclic peptides, as well as Ac-WFW, was dominated by enthalpy changes

(Table 6). One exception is the binding of c-(Bal)F(Bal) to POPC/LA, which is characterized

by comparable contributions of H° (–15.4 kJ/mol) and –TS° (–18.31 kJ/mol) to the free

energy of binding. Comparable data were derived for c-(Bal)F(Bal) binding to mixed

POPC/POPG bilayers. Another interesting observation is that Ac-WFW, the flexible b3-hW-

peptide, and the two most hydrophilic cyclic peptides show little change in H° and –TS°

values with variation in the LPS-moiety (Table 6). In contrast, the contribution of enthalpy to

binding of the hydrophobic peptides increases whereas the entropic effect decreases.

Unlike the expectation of the differentiated activity pattern against E. coli [70], no

dependency of the G° to s-LPS-doped bilayers was observed upon the composition of the

cyclic peptide, the only exception was c-(5fW)F(5fW). The aromatic residue is characterized

by a reduced quadrupole and enhanced dipole moment and an increased hydrogen bonding

ability compared to W (Table 2). However, enhanced binding does not improve the activity

(Table 4). The peptide is as active as the parent peptide c-WFW against E. coli.

3.1.5.4 Effect of ionic strength upon c-WFW binding to r-LPS and s-LPS

lipid systems

To investigate whether the enhanced partitioning of the cyclic peptides into LPS-

doped POPC bilayers is due to electrostatic interactions, titrations of c-WFW with POPC/r-


43

LPS (12/1 [mol/mol]) and POPC/s-LPS (12/1 [mol/mol]) were performed at different NaF

concentrations. The binding isotherms are shown in Fig. 23 and the thermodynamic

parameters are presented in Table 7. The increasingly exothermic enthalpy change with

enhanced ionic strength was compensated by an increasingly unfavorable entropic

contribution.

As observed previously, binding was strongest to bilayers containing O-antigen-

presenting s-LPS. For c-WFW interacting with POPC/r-LPS, only a slight influence of the

ionic strength on K0 was found. By contrast, peptide binding to POPC/s-LPS was slightly

dependent upon ionic strength: K0 decreased from 2.7 × 10–4 to 1.6 × 10–4 M–1 upon

increasing the salt concentration from 75−200 mM. The low salt dependency of c-WFW

binding to POPC/r-LPS bilayers confirms a minor role of direct electrostatic interaction of the

peptide with LPS inner core charges (Table 7, Fig. 23). This is related to a low antimicrobial

activity observed for cyclic RW-peptides against E. coli mutant strains with outer core- and

O-antigen deficient-LPS [70].

Fig. 23. Isotherms for c-WFW binding to r-LPS- and s-LPS- bearing POPC SUVs at different ionic

strengths (T = 37 C). The salt concentrations (cNaF) were (●) 75 mM, (▲) 125 mM, (♦) 200 mM for POPC/r-

LPS (12/1 [mol/mol]) and (○) 75 mM, ( ) 125 mM, (◊) 200 mM for POPC/s-LPS (12/1 [mol/mol]) lipid bilayers in 10 mM phosphate buffer, pH 7.4.


44

Table 7. Salt dependency of the thermodynamic parameters for binding of c-WFW to POPC/r-LPS (12/1 [mol/mol]), and POPC/s-LPS (12/1 [mol/mol]) lipid bilayers in 10 mM phosphate buffer, pH 7.4 (T = 37 C).

Thermodynamic parameters

Lipid bilayer SUV* cNaF (mM)

H°

(kJ/mol)

TS°

(kJ/mol)

G°

(kJ/mol)

K0 (M-1) zc-WFW

POPC/r-LPS 75 25.3 7.8 33.1 6.9 × 103 1.6

125 28.4 4.0 32.4 5.2 × 103 1.9

200 31.0 0.7 31.7 4.0 × 103 1.8

POPC/s-LPS 75 27.6 9.0 36.6 2.7 × 104 0.8

125 35.0 0.1 35.1 1.5 × 104 0.9

200 41.0 5.7 35.3 1.6 × 104 1.2 *The peptide and lipid concentrations were 40 M and 5 mM, respectively.

The high degree of peptide binding to POPC/s-LPS, compared to POPC/r-LPS, has to

be associated with the presence of outer core and O-antigen oligosaccharides. The fact that the

partitioning pattern of the cyclic peptides revealed little variation points to a dominating

influence of electrostatic interactions upon binding (Figs. 22A and 23). Both r-LPS and s-LPS

have the same distribution of negatively charged phosphate groups; however, unlike r-LPS,

bilayer binding became more salt dependent for POPC/s-LPS (Fig. 23). The presence of the

sugar moieties seems to favor the electrostatic component in binding. As a result of enhanced

accumulation, peptide partitioning might increase.

3.1.5.5 Heat capacity change on membrane partitioning of c-WFW

A common feature of both classical and nonclassical hydrophobic effects is their

strong temperature dependency, which is characterized by negative Cp° values [138]. To

determine the heat capacity of peptide-lipid interactions, Cp°, the partitioning of c-WFW into

SUVs composed of POPC or POPC/POPG (3/1 mol/mol) as a function of temperature was

measured.

The results are summarized in Fig. 24 and Table 8. The H° values vary almost linearly and

become more exothermic with increasing temperature, as reflected by large negative Cp°

values of –182 J/(K mol) and –118 J/(K mol) for POPC and POPC/POPG (3/1 [mol/mol])

respectively. This signature of the hydrophobic effect results from the release of ordered


45

water molecules from hydrophobic surfaces upon partitioning of the peptide into the lipid

bilayer [138]. Another contribution to Cp° may come from an increase in lipid acyl chain

motion induced by membrane expansion on peptide binding [131]. These results are in

contrast to large positive Cp° values found for membrane partitioning of magainins [139].

Fig. 24. Temperature dependency of the binding enthalpies of c-WFW to (□) POPC and (○) POPC/POPG (3/1 [mol/mol]) SUVs.

Table 8. Temperature dependency of the thermodynamic parameters of binding of c-WFW to POPC and POPC/POPG (3/1 [mol/mol]) lipid bilayers.

Thermodynamic parameters

Lipid vesicles Temp.

(C)

H°

(kJ/mol)

TS°

(kJ/mol)

G°

(kJ/mol)

K0 (M-1) Cp

°

(J/K mol)*

12 26.6 7.6 34.2 3.4 × 104

25 27.2 6.7 33.9 1.6 × 104 182.1

POPC (SUVs)

37 31.2 1.8 33.0 6.7 × 103

12 21.2 13.5 34.7 4.1 × 104

25 21.8 13.0 34.8 2.3 × 104 118.1

37 23.5 11.0 34.5 1.2 × 104

POPC/POPG (SUVs)

50 25.6 9.2 34.8 7.8 × 103 *The values were determined by linear regression of the experimental values presented in Fig. 24 (H° vs. T).


46

3.1.6 Summary

Induction of conformational constraints in small RW-rich peptides by cyclization has

been found to distinctly enhance antimicrobial activity, in particular against Gram-negative

E. coli [53,68]. RW-rich clusters seem to be responsible for the selectivity increase as well as

for the high affinity to lipid bilayers [53], and LPS moieties have been suggested to exert a

strong activity-modulating effect [70].

In this study, W residues in c-WFW were replaced by various unnatural amino acids,

such as Dht, Igl, 5MeoW, 5fW, b3-hW, 5MeW, 1MeW, and Bal (Fig. 13). This made possible

a systematic investigation of aromatic clusters that affect peptide interactions with lipid

systems mimicking the outer and inner membranes as well as of the biological activities

against E. coli in comparison with Gram-positive B. subtilis and RBCs.

This study showed that:

i. Peptide hydrophobicity and backbone constraints are the crucial parameters for the

biological activity (Hydrophilic and the linear and large cyclic sequences are least

active) (Table 4).

ii. Compared to B. subtilis, the activity profile against the E. coli strain is much more

differentiated (Table 4).

iii. E. coli is slightly less susceptible than B. subtilis to the cyclic peptides and the

hemolytic activity of the cyclic peptides is low (Table 4).

iv. The activity profile against bacteria correlates with the profile of free energy of

partitioning into phospholipid bilayers (Fig. 21 and Table 4). The negative charge in

POPC/POPG bilayers enhances Kapp. but K0 is independent upon the presence of the

anionic lipid (Fig. 20 and Table 5).

v. Low partitioning into POPC and mixed POPC/POPG bilayers correlates with low

hydrophobicity of the cyclic peptides, the large cycle containing -amino acid, and the

linear sequence (Fig. 21 and Table 5).

vi. Partitioning into POPC/LA bilayer is comparable to partitioning into

POPC/POPG systems (Fig. 22 and Table 6).


47

vii. Sugar moieties of r-LPS and s-LPS modify the partitioning behavior. Lipid

interactions are strongest for POPC doped with s-LPS. The difference between the

linear and cyclic peptides is conserved; however, partitioning does no longer depend

on the structural properties of the cyclic sequences. The results clearly demonstrate a

strong supporting role of the outer core and O-antigen in peptide binding to LPS rich

bilayers (Fig. 22 and Table 6).

Membrane permeabilization by many cationic peptides depends upon both

electrostatic accumulation by negatively charged membrane constituents and insertion into the

bilayer, which finally results in a breakdown of the barrier function of the lipid matrix [44].

This idea is supported by the correlation of ITC binding data with hemolytic and antibacterial

activities of c-WFW and its analogs. Accordingly, low peptide concentrations in the vicinity

of neutral lipid bilayers are responsible for low hemolytic activities, whereas electrostatic

attraction to negatively charged membranes favors partitioning into POPC/POPG bilayers and

POPG-rich (~70 mol%) B. subtilis membranes [23]. In addition to the effect of the outer

membrane, the slightly reduced susceptibility of E. coli could be due to lower peptide

accumulation at the inner target membrane, which contains only 15 mol% negatively charged

lipids [23]. However, the hydrophobic contributions to binding (as expressed by K0; see

Table 5) were identical. Peptide hydrophobicity and backbone constraints were found to be

the crucial determinants of biological activity.

A balance between electrostatic and hydrophobic contributions to peptide-lipid

interactions has been suggested to determine the positioning of RW-rich hexapeptides in the

bilayer interface [68]. The preferential location of W residues at the membrane interface has

been attributed to their aromaticity and ability to form hydrogen bonds with both water and

polar lipid headgroup moieties [58,63,136,137,140,141]. In this study, hydrophobicity and

conformational flexibility of RW-rich hexapeptides were identified as the crucial parameters

which affect binding. This investigation confirmed the suggestion that peptide interactions

with the cytoplasmic membrane determine the biological effect. Other modifications in the

hydrophobic cluster of the cyclic hexapeptides have only a minor influence upon peptide

interaction with biological systems and model membranes.


48

Strong peptide interactions with the outer-membrane LPSs of E. coli influence the

peptide transport across the outer wall and thus the accessibility at the target membrane. The

affinities of the hexapeptides to POPC/LA and POPC/POPG bilayers, with identical negative

surface charge densities, are comparable with respect to the magnitude and sequence

dependency of the thermodynamic parameters. While interactions with the LA domain were

not particularly strong, peptide partitioning was favored into POPC/r-LPS and even more

pronounced in the presence of s-LPS. This behavior correlates with the reduced antimicrobial

activity against O-antigen- and outer-core-deficient LPS mutant E. coli strains [70] and

confirms the activity-modulating role of the E. coli outer wall. On the basis of these

observations, it is concluded that hydrophobic peptide-LPS interactions are essential for an

efficient transport across the bacterial outer membrane, but that LA does not act as a specific

activity-modulating binding site for the hexapeptides. It seems that the O-antigen of LPS is

essential for avid partitioning and likely decisive for efficient peptide transport across the

E. coli outer wall.


49

3.2 Site-specific immobilization of CAPs

In order to investigate the suitability of CAPs for the generation of antimicrobial

surfaces, and to analyze parameters such as: the distance between the active sequences and the

solid matrix; the density of peptide-loaded surface and the surface area available for peptide

loading, which may influence the activities of tethered peptides upon bacterial cells and lipid

bilayers; synthesis resins, i.e., TentaGel S NH2, TentaGel M NH2, TentaGel MB NH2,

HypoGel 400 NH2, and HypoGel 200 NH2 were used as models of solid surfaces. The

variability of resin beads in size, capacity and spacer length represent a set of critical

parameters to be analyzed. The model KLAL peptide and the MAG 2-derived MK5E, two

highly membrane active -helical CAPs, were used for this purpose. The model KLAL

peptide has both antimicrobial and hemolytic activity [44], while MK5E is selectively active

against bacteria [118]. KLAL peptide was suggested to act via a carpet-like mode of action

[44] whereas MAG 2, the parent peptide of MK5E, was described to form toroidal pores on

interaction with bacterial cells and model membranes [118].

The surface tethering of CAPs is known to reduce the flexibility of peptides and may

limit the range of peptide penetration to bacteria cytoplasmic membrane. Due to different

mechanisms being responsible for the antimicrobial effect of the peptides, site-specific

tethering is expected to be used as a tool to get information on the mode of action of CAPs

(Fig. 7). To examine this hypothesis, KLAL and MK5E peptides, as well as three natural

CAPs (i.e., MEL, TP, and BUF), were tethered at C terminus, N terminus, and side chains via

an -amino group of K residues. MEL, BUF, and TP form amphipathic structures at peptide-

lipid interfaces. MEL was suggested to act on bacterial cells through pore formation [8],

whereas BUF targets intracellular processes after translocation across the cytoplasmic

membrane [46]. TP has ambiguous mechanisms of action: membrane depolarization coupled

to secondary intracellular targeting [119,120].

The antimicrobial activities of tethered and soluble peptides were correlated to their

lipid bilayers’ permeabilizing activities, not only in order to identify critical parameters in

desigining effective biocidal surfaces, but also to examine their applications in order to shed

light on to the modes of action of CAPs.


50

3.2.1 Physical and chemical properties of PEGylated resins as

model solid surfaces

Synthesized resins, with PEG spacers of different length, were used to couple the

peptides at different chain positions. PEG has been described to provide interfacial protective

coating [142] and thus to improve the applicability (solubility and stability against enzymatic

degradation) of proteins [143] and peptides [144].

TentaGel S NH2, TentaGel M NH2, TentaGel MB NH2, HypoGel 400 NH2, and

HypoGel 200 NH2 belong to the classes of divinyl benzene cross-linked polystyrene

containing PEG grafts (Fig. 25). They are highly porous. The size of the pores for a

TentaGel S NH2 resin bead ranged between 0.10.2 m, which is smaller than the size of a

bacterium [145]. The PEG grafts have different spacer lengths and represent the majority of

the mass of these polymers. Thus, the properties of these hybrid resin beads closely resemble

those of PEG [146]. Furthermore, the reactive centers are located at the terminus of the glycol

spacers. These properties provide the opportunities for synthesizing combinatorial libraries

using organic solvents followed by bioassays in aqueous media [147,148]. These resins have

various size distributions ranging from TentaGel M NH2 (diameter 10 m) to

TentaGel MB NH2 (diameter ~ 300 m) (Table 9). Whereas TentaGel S NH2 is characterized

by a long spacer (3 kDa) and has the lowest capacity, HypoGel 400 NH2, and

HypoGel 200 NH2 have much shorter PEG spacers and comparably high capacities.

Fig. 25. Basic chemical structure of TentaGel S NH2, TentaGel M NH2, TentaGel MB, HypoGel 400 NH2 and HypoGel 200 NH2. P stands for the resin bearing PEG, and n represents the number of ethylene oxide units.


51

Table 9. Physical and chemical characteristics of TentaGel S NH2, TentaGel M NH2, TentaGel MB NH2, HypoGel 400 NH2, and HypoGel 200 NH2.

Resin Capacity (mmol/g)

Diameter (m)

Beads/g* Capacity/Bead (pmol)*

MW of spacer (Da)

n (ethylene oxide units)

TentaGel S NH2 0.32 130 8.87 × 105 280−330 3000 75

TentaGel M NH2 0.25 10 1.95 × 109 0.13 NA† NA

TentaGel MB NH2 0.26 ~ 300 6.40 × 104 4000 NA NA

HypoGel 400 NH2 0.69 110−150 NA NA 400 10

HypoGel 200 NH2 0.92 110−150 NA NA 200 5 *These data are taken from the ref. [145].

†NA, not available.

3.2.2. Activity of surface tethered membrane-active CAPs - role of

tethered peptide site

3.2.2.1 Characterization of KLAL and MK5E peptides by HPLC and CD

N-terminally acetylated KLAL and MK5E peptides, as well as sequences bearing

PEG 2 chains at the intended positions of immobilization, were prepared to assess the

influence of immobilization related charge modification and introduction of the PEG moiety

on the peptide structure and biological effect. Table 10 shows the PEGylated and acetylated

KLAL and MK5E peptides, their helicity and the tR-HPLC values as a measure of

hydrophobicity.

Introduction of the hydrophilic PEG 2 chain did not substantially influence the

retention behavior. This might be due to the low molecular mass (163 Da) of PEG 2. In

contrast, N-terminal acetylation caused reduction of positive peptide charge and this increase

in hydrophobicity also enhanced the tR.

CD spectroscopic investigations of KLAL, MK5E and the acetylated and PEGylated

analogs confirmed an unordered peptide conformation in phosphate buffer (Fig. 26). TFE

induced -helical conformation, as reflected by negative ellipticities at 207 nm and 222 nm

and a positive CD band below 200 nm (Fig. 26). Although, the helical content of MK5E and

Ac-MK5E was quite similar (~ 50%), the helicity of the most hydrophobic Ac-KLAL was

enhanced by more than 30% compared with KLAL (Table 10). PEGylation decreased the


52

peptide helicity independent of the position of PEG introduction (Table 10). Steric hindrance

might be responsible for this effect.

Table 10. Amino acid sequences, abbreviations, tR-RP-HPLC and helicity of peptides.

NO Denotation Peptide sequence Calculated helicity

tR (min)

[] %

1 KLAL KLALKLALKALKAALKLA-NH2 20.6 52

2 Ac-KLAL Ac-KLALKLALKALKAALKLA-NH2 24.0 68

3 PEG-KLAL* PEG-KLALKLALKALKAALKLA-NH2 21.9 44

4 KLAL-PEG KLALKLALKALKAALKLA-PEG-NH2 20.3 43

5 Ac-PEG-KLAL Ac-PEG-KLALKLALKALKAALKLA-NH2 23.7 51

6 Ac-KLAL-PEG Ac-KLALKLALKALKAALKLA-PEG-NH2 23.8 43

7 Ac-KLAL5PEG Ac-KLALK(PEG)LALKALKAALKLA-NH2 24.4 48

8 Ac-KLAL9PEG Ac-KLALKLALK(PEG)ALKAALKLA-NH2 24.2 41

9 MK5E GIGKFIHAVKKWGKTFIGEIAKS-NH2 18.7 50

10 Ac-MK5E Ac-GIGKFIHAVKKWGKTFIGEIAKS-NH2 20.9 51

11 PEG-MK5E PEG-GIGKFIHAVKKWGKTFIGEIAKS-NH2 19.4 29

12 MK5E-PEG GIGKFIHAVKKWGKTFIGEIAKS-PEG-NH2 18.7 29

13 MK5E10PEG GIGKFIHAVK(PEG)KWGKTFIGEIAKS-NH2 18.9 ND†

14 Ac-PEG-MK5E Ac-PEG-GIGKFIHAVKKWGKTFIGEIAKS-NH2 20.7 30

15 Ac-MK5E-PEG Ac-GIGKFIHAVKKWGKTFIGEIAKS-PEG-NH2 20.9 29

16 Ac-MK5E10PEG Ac-GIGKFIHAVK(PEG)KWGKTFIGEIAKS-NH2 21.8 ND *PEG, bifunctional PEG 2 (2 ethylene oxide units).

†ND, not determined.

Fig. 26. CD spectra of model KLAL peptide and MK5E, and their acetylated analogs in (A) phosphate buffer and (B) TFE/buffer (1/1 [v/v]) (T = 25 C). The peptides are presented as KLAL (red),

Ac-KLAL (pink), MK5E (blue), and Ac-MK5E (turquoise).


53

3.2.2.2 Preparation and characterization of tethered KLAL and MK5E

peptides

C-terminally immobilized KLAL and MK5E on TentaGel S NH2, HypoGel 200 NH2,

and HypoGel 400 NH2 were prepared by standard SPPS using Fmoc-amino acids [149]. It is

known from the literature [101] that the major sources of impurity in SPPS are deletion

sequences and chain termination that might occur at each coupling step due to non-

quantitative coupling reactions. Thus, the activities of C-terminally immobilized peptides

might result not only from the right sequences, but also from minor contributions of deficient

sequences. N terminus and side chain immobilization of KLAL and MK5E on

TentaGel S NH2 using the thioalkylation [150] and oxime-forming ligation strategies [151]

were performed with HPLC-purified sequences (Fig. 27). Thus, the antimicrobial activities of

these immobilized peptides result only from the correct sequences.

Fig. 27. Chemical strategies for side chain, C terminus and/or N terminus immobilization of CAPs. (A) Oxime-forming ligation, (B) Thioalkylation.

The formation of tethered peptides was confirmed after cleavage of the sequences

immobilized on TentaGel S RAM resin (Fig. 28). TentaGel S RAM resin is similar in the


54

chemical structure to TentaGel S NH2 resin, but has a TFA cleavable linker. The molecular

masses of the peptides coupled via the thioalkylation and oxime formation were 57 Da. and

69 Da. larger than those values for the Cys- and AOA-modified peptides, respectively

(Fig. 29).

Fig. 28. The chemical strategy applied to confirm the formation of the tethered peptides. The tethered peptides are released from TentaGel S RAM after treatment with TFA and are characterized by analytical-HPLC

and LC / ESI-TOF MS.

m/z200 400 600 800 1000 1200 1400 1600

%

0

679.7520

679.4196

510.0711299.9843

680.0984

1019.13731018.6370

680.4309

680.7567

1019.6462

1020.12981076.6631

A 100

m/z200 400 600 800 1000 1200 1400 1600

%

0

886.4650

299.9848

308.9922

886.0854359.9636665.3541

886.4808

886.8289

1329.2207924.4839924.7909 1329.7535

B100

Fig. 29. LC-MS spectra of N terminus tethered (A) KLAL and (B) MK5E. The ligation products result from the reaction between the N-terminally Cys- or AOA-modified KLAL and MK5E and TentaGel S RAM pretreated with BrAcOH and pyruvic acid, respectively. The signals represent the mass values for the

thioalkylation and ligation products at various m/z. Mass differences in the peaks reflect TFA addition to the peptides during electrospray ionization. (TFA is used as an additive in the mobile phase for LC / ESI-TOF MS)

(A) [M+2H+TFA]2+ = 1076.66, [M+2H]2+ = 1018.64, [M+3H]3+ = 679.42, [M+4H]4+ = 510.07. (B) [M+2H]2+ = 1329.22, [M+3H+TFA]3+ = 924.48, [M+3H]3+ = 886.09, [M+4H]4+ = 665.35.


55

A comparison of the resin capacity and the density of immobilized peptides shows that

about 1/3 of the reactive groups of resins was occupied with C-terminally immobilized

peptides (Tables 12, 13). Furthermore, under the applied coupling conditions, the density of

C-terminally TentaGel S NH2-bound peptides (0.099 and 0.133 mol/mg for KLAL and

MK5E peptide respectively) was about three times higher than the density of the N-terminally

and side chain-tethered sequences. This might be partially due to the different synthesis

procedures and limited accessibility of the reactive functional groups. It has been reported that

at most 15% of the total amount of functional groups of typical TentaGel beads are located on

the surface of the bead [152]. Moreover, protein immobilization onto TentaGel was shown to

be limited to the bead surface [153]. Whereas SPPS on the porous resin will result in a

substantial amount of peptides, which might be not accessible for interaction with biological

membranes, immobilization of the complete peptide sequences via thioalkylation and ligation

strategies will be restricted to the surface of the beads. The enhanced capacity of the

HypoGels (Table 9) leads to an enhanced (Table 13) but, compared to TentaGel S NH2, the

coupling efficiency slightly decreased as reflected by an increase in the ratio of resin capacity

to peptide density.

3.2.2.3 Biological activities of free and tethered KLAL and MK5E peptides

Antimicrobial activity of the free peptides

All KLAL and MK5E peptides showed activity against B. subtilis and E. coli in the

micromolar range (Table 11).

To model the loss of charge connected with immobilization via the N-terminal -

amino group, acetylated and N-terminally free KLAL and MK5E sequences were compared.

The activity of the KLAL peptides was highest against B. subtilis and independent of

chemical modification with PEG 2 (MIC 0.8 M). As a consequence of charge reduction,

the antibacterial activity of acetylated KLAL peptides against E. coli was 4−16 fold reduced

compared with the parent sequence, whereas changes in the activity against B. subtilis were

not observed. MK5E and Ac-MK5E were similarly active against B. subtilis (MIC 1.6 M),

but acetylation reduced the anti-E. coli activity of the peptide. The loss of one cationic charge

distinctly enhanced the hydrophobicity of the peptides as was reflected by an increase in tR-

HPLC (Table 10). Likely, the tR value of Ac-KLAL was further enhanced by an increased

amphipathicity, which is based on the enhanced helicity at interfaces as has been described for


56

other peptides [154], and found here for Ac-KLAL as compared with KLAL under structure

inducing solvent conditions.

Table 11. Antimicrobial and hemolytic activities of KLAL and MK5E peptides and their PEGylated analogs against B. subtilis and E. coli bacteria and RBCs.

Peptide Antimicrobial activity* Hemolytic activity

MIC (M)† EC25 (M)

NO Denotation B. subtilis (DSM 347) E. coli (DH 5α) Erythrocyte lysis

1 KLAL 0.8 1.6 13.1

2 Ac-KLAL 0.8 12.5 9.1

3 PEG-KLAL 0.8 3.1

4 KLAL-PEG 1.6 1.6

5 Ac-PEG-KLAL 0.8 12.5

6 Ac-KLAL-PEG 0.8 6.3

7 Ac-KLAL5PEG 0.8 25.0

8 Ac-KLAL9PEG 0.8 12.5

9 MK5E 1.6 0.8 334.0

10 Ac-MK5E 1.6 3.1 > 400.0

11 PEG-MK5E 1.6 1.6

12 MK5E-PEG 1.6 1.6

13 MK5E10PEG 1.6 1.6

14 Ac-PEG-MK5E 6.3 12.5

15 Ac-MK5E-PEG 6.3 6.3

16 Ac-MK5E10PEG 3.1 6.3 *The results are the mean of three independent experiments performed in triplicate with a standard deviation of less than 5% (Fig. 39).

†MIC values were determined after 17 h incubation at 37 C. MBC MIC.

Although the introduction of PEG 2 at different positions of both KLAL and MK5E

caused the antimicrobial activities and the helicity of the N-terminally free and acetylated

parent sequences to decease slightly (differences of one dilution step among the antimicrobial

activities of PEGylated KLAL and MK5E peptides), changes in the activity spectrum were

not observed (Tables 10, 11). One might speculate that these small changes are due to the low

molecular weight of the attached PEG 2 moiety. However, recently it has been reported that

even coupling of much larger PEG units did not influence the basic mechanism of membrane

permeabilization of amphipathic peptides [155,156]. Thus, modification of MG 2 with PEG


57

(5 kDa) resulted only in slightly reduced antimicrobial activity and did not change the

interaction patterns with lipid bilayers [155]. Similar observations were made with β-sheet

tachyplesin [156]. In contrast, the loss of the antimicrobial activity of PEG-nisin [144] could

only be explained by the disturbance of the peculiar mechanism of action, which is based on

selective lipid II binding and consecutive migration of the peptide’s C terminus through the

cell membrane.

These results show that the loss of the N-terminal charge may result in a drastic

reduction of the antimicrobial activity, in particular against Gram-negative bacteria, whereas

introduction of PEG chains at different chain positions might be expected to have little

influence. Therefore, it is concluded that the conservation of the cationic charge with peptide

immobilization is particularly important for maintaining the peptide activity against Gram-

negative bacteria.

Antimicrobial activity of the tethered peptides

Peptides immobilized by the thioalkylation and ligation strategies inhibited the growth

of both bacteria at similar MIC regardless of the position of immobilization (Table 12).

Concentrations between 0.1−0.2 mM, and 0.6−0.8 mM of KLAL peptides immobilized at the

N terminus and side chains were required to inhibit bacterial growth and to act bactericidally

against B. subtilis and E. coli, respectively (Table 12). The activity of randomly immobilized

KLAL was comparable to other side chain-specific immobilized sequences.

The activity profile of TentaGel S NH2-bound MK5E peptides was found to be

slightly different from KLAL (Table 12). The side chain and C-terminally immobilized

MK5E sequences showed comparable activities, but a charged N-terminal -amino group

seems to be necessary for maximal bactericidal activity in particular againat E. coli, which

diminish with either acetylation or N-terminal immobilization. The comparably high MIC

values of C-terminally immobilized KLAL and MK5E are likely related to the fact that the

majority of these sequences (~ 75%; see ref. [152]) were tethered in the interior of the porous

TentaGel S NH2 and might not be accessible for membrane interaction [152,153]. Indeed, the

small pore size of TentaGel S NH2 [145] does not allow for bacterial entry and penetration. In

all cases, the MBC values of the immobilized peptides were identical to the MIC.


58

Table 12. The antimicrobial activities of KLAL and MK5E peptides immobilized on TentaGel S NH2 resin against B. subtilis and E. coli along with the densities of resin-immobilized peptides and the MICs of the corresponding PEGylated free peptides.

Antimicrobial activity of immobilized peptides

MIC

B. subtilis (DSM 347) E. coli (DH 5)

Peptide Position of immobilization

Density of peptide

(mol/mg)* Resin

(mg/ml)† Immobilized

(mM)‡ Resin

(mg/ml)† Immobilized

(mM)‡

KLAL C terminus 0.099 2 0.20 25 2.47

N terminus 0.028 5 0.14 25 0.70

K 5§ 0.024 5 0.12 25 0.60

K 9 0.028 2 0.06 25 0.70

K 12 0.030 5 0.15 25 0.75

Random 0.031 5 0.15 25 0.77

Ac-KLAL C terminus 0.099 2 0.20 45 4.45

MK5E C terminus 0.133 5 0.67 5 0.67

N terminus 0.026 10 0.26 15 0.39

K 4 0.039 ND¶ ND 5 0.19

K 10 0.033 5 0.17 5 0.17

K 14 0.025 5 0.13 5 0.13

Ac-MK5E C terminus 0.133 10 1.33 45 5.99 *The amount of resin-immobilized peptides was determined from three independent experiments based on the absorption of the Fmoc-chromophore at 301 nm ( = 6000 M-1 cm-1). The standard deviations ranged between 2 and 10 %.

†Each MIC of peptide-covered resin was determined in one experiment using a serial dilution of the peptide-loaded resin. The values were confirmed in two independent experiments using the determined MIC and two resin concentrations below and above the MIC. No changes were found (Fig. 40).

‡The MIC of the immobilized peptides was calculated on the basis of the concentration of peptide-covered resin causing growth inhibition and taking into consideration the density of the peptides on the resin beads. Based on the standard deviation of the surface density, variations in the MIC are less than 10 %.

§The numbers give the chain position of immobilization. K stands for the lysine residue.

¶ND, not determined

Consequently, tethering conserved the activity spectra of the peptides at reduced

concentrations. The resin-bound peptides were antimicrobial against E. coli and B. subtilis in

the millimolar range compared to the results seen with micromolar concentrations of the free

peptides. Moreover, insertion of either KLAL or MK5E into the membrane as a mode of

action is supported by the fact that the activity is independent of the site of immobilization. As

the immobilization at TentaGel S NH2 was performed at the chain termini and the K residues


59

in the polar face of peptide helices, the immobilized peptides are assumed to arrange their

amphipathic helix in the membrane surface.

Hemolytic activity of the free and tethered peptides

KLAL peptides were active towards RBCs with EC25 values of about 10 M

(Table 11). MK5E was not hemolytic up to concentrations of about 400 M. TentaGel S NH2

showed a concentration dependent hemolytic effect. Hemolysis was less than 10% up to about

80 mg/ml, but rapidly increased at higher concentrations (Fig. 30). The activities of 40 mg

and 80 mg TentaGel S NH2-bound KLAL, Ac-KLAL, MK5E and Ac-MK5E were not

distinguishable from the activity of the bare resin beads. This observation leads to the

conclusion that at their MICs (amount of resin < 45 mg/ml; Table 12) both immobilized

KLAL and MK5E peptides are inactive towards RBCs.

Fig. 30. The hemolytic activity of TentaGel S NH2 resin beads.

3.2.2.4 Bilayer permeabilizing activities of free and tethered KLAL and

MK5E peptides

The ability of peptides to permeabilize the lipid bilayer of liposomes and thus to

induce the release of incorporated dye was measured in order to compare the membrane

permeabilizing activity of free and tethered peptides. The measurements were to provide

information about the peptide interaction with lipid matrices of variable composition as

physical models of biological targets. Electrically neutral POPC LUVs and mixed vesicles,

i.e, POPC/POPG (1/3 [mol/mol]) and POPC/POPG (3/1 [mol/mol]) were employed in order


60

to mimic the charge properties of the lipid matrix of RBCs, the membrane of Gram-positive

and the inner membrane of Gram-negative bacteria respectively.

The free peptides

The EC50 values of initial calcein leakage (Fig. 31) showed that all acetylated and

PEGylated peptides permeabilize highly negatively POPC/POPG (1/3 [mol/mol]) LUVs in a

narrow micromolar concentration range. With reduction of the negative bilayer charge, the

peptide activity became more differentiated. The activity of all KLAL and Ac-KLAL peptides

distinctly increased (EC50 decrease) with reduction of anionic bilayer charge. In contrast, the

activity of MK5E and Ac-MK5E was only slightly modified with variation in the lipid

composition and the PEGylated peptides showed enhanced EC50 values with decreasing the

POPG content of liposomes. This activity reduction was most obvious for Ac-MK5E, but

little dependent upon the PEG position. Interestingly, the surface affinity and the

permeabilizing effect upon lipid membranes correlated well with the antimicrobial activity

profiles (Table 11, Fig. 31).

Fig. 31. The bilayer (LUVs) permeabilizing activity of KLAL and MK5E peptides. The vesicles are presented as POPC (black), POPC/POPG (3/1 [mol/mol]) (white), and POPC/POPG (1/3 mol/mol) (gray). EC25

was determined at cL 25 M in buffer after 1 min.


61

The tethered peptides

The kinetics of the dye release from POPC, and mixed POPC/POPG LUVs induced by

C-terminally TentaGel S NH2-bound KLAL and MK5E peptides is presented in Fig. 32.

Although different in the magnitude, each of the resin-tethered peptides induced disruption of

the bilayers in a dose-dependent manner. The activity profile of the bound peptides correlated

well with the effects of the corresponding free peptides. Non-modified TentaGel S NH2 was

not bilayer active. These results suggest that C-terminal immobilization via a large PEG chain

conserved the bilayer permeabilizing ability of the free KLAL and MK5E peptides.

Fig. 32. Kinetics of dye release from (A) POPC, (B) POPC/POPG (3/1 [mol/mol]), and (C) POPC/POPG (1/3 [mol/mol]) LUVs induced by tethered peptides. The tethered peptides are C-terminally

immobilized at TentaGel S NH2 and presented as KLAL (red), Ac-KLAL (pink), MK5E (blue), and Ac-MK5E (turquoise). The final concentrations of tethered peptides are as follows: for POPC: KLAL (0.8 mM), Ac-

KLAL (0.8 mM), MK5E (1.1 mM), and Ac-MK5E (1.1 mM); for POPC/POPG (3/1 [mol/mol]): KLAL (1.6 mM), MK5E (2.1 mM); and for POPC/POPG (1/3 [mol/mol]): KLAL (0.8 mM), MK5E (1.1 mM).

The dye release was monitored as increase in the fluorescence intensity at 514 nm at cL 25 M.


62

3.2.3 Influence of physical characteristics of solid surfaces upon

biocidal activity

3.2.3.1 Effect of spacer length

Because the thickness of the cell envelope for E. coli and B. subtilis has been reported

to be 46 nm and 45−55 nm respectively [157−159], the role of the spacer length upon the

antimicrobial activities of KLAL and MK5E peptides C-terminally immobilized on

TentaGel S NH2, HypoGel 400 NH2 and HypoGel 200 NH2 was investigated. The data

summarized in Tables 12 and 13 illustrate that peptide coupling via a long PEG spacer

(TentaGel S NH2) resulted in high antimicrobial activity against both strains whereas the PEG

spacers of HypoGel 200 NH2 and HypoGel 400 NH2 are too short (Table 9) to span the highly

negatively charged LPS-rich wall of Gram-negative and the peptidoglycan layer of Gram-

positive bacteria. The biocidal activities were as follows:

TentaGel S NH2 > HypoGel 400 NH2 > HypoGel 200 NH2. Peptides attached via the long

TentaGel S NH2 spacer can interact with the cytoplasmic membrane, which is the target for

antimicrobial peptides [1]. The observation suggests that a long spacer-related high flexibility

of the immobilized peptide is of advantage for antimicrobial activity.

Table 13. Antimicrobial activities against B. subtilis and E. coli bacteria and peptide densities of C-terminally immobilized KLAL and MK5E peptides on HypoGel 400 NH2, and HypoGel 200 NH2.

Antimicrobial activity (HypoGel 400 NH2 / HypoGel 200 NH2)

B. subtilis (DSM 347) E. coli (DH 5) Peptide Density of peptide

(mol/mg) MIC Resin

(mg/ml) MIC Peptide

(mM) MIC Resin

(mg/ml) MIC Peptide

(mM)

KLAL 0.151 / 0.247 10 / 15 1.51 / 3.71 55 / 70 8.31 / 17.29

Ac-KLAL 0.151 / 0.247 15 / 15 2.27 / 3.71 75 / >80 11.33 / >19.76

MK5E 0.180 / 0.253 10 / 15 1.80 / 3.79 15 / 10 2.70 / 2.53

Ac-MK5E 0.180 / 0.253 20 / 20 3.60 / 5.06 65 / 60 11.70 / 15.18

3.2.3.2 Surface density of tethered peptides

The bacterial membrane possesses in the order of 105 anionic charges [160]. Taking into

consideration that a Gram-negative E. coli has an average surface area of 0.5 × 2.0 m2, the

membrane charge density is approximately 1013 charges/cm2. The capacity of TentaGel S NH2


63

is 280−330 pmol/bead (Table 9). Taking KLAL and MK5E peptides, the density of positive

surface charges per TentaGel S NH2 bead is much higher than the surface charge density of an

E. coli bacterium, even if only one part of the total charge of the resin (at least

~ 2 × 1017 charges/cm2) is considered. Therefore, the peptide density on the surface of

TentaGel S NH2 beads is sufficient to promote resin-cell interaction as demonstrated by the

fact that higher loading of the HypoGels did not enhance the activity (Tables 12, 13).

Moreover, the peptides loading on HypoGel 200 NH2, which is about twice of the peptides

density on HypoGel 400 NH2 (Table 13), showed that increasing the peptide loading did not

improve the biocidal activity if the flexibility of the immobilized peptide is limited by a short

spacer. These results lead to the suggestion that with increased constraints induced by a

reduced spacer length, the ability of peptides to bind to the bacterial membrane was

conserved; however, the membrane permeabilization efficiency, e.g. the ability of the peptides

to insert into the target membrane was reduced. Nevertheless, the antimicrobial activity of

HypoGel-bound peptides suggests that interactions with the outer layer of bacteria provide a

substantial contribution to the effect. For cationic biocidal polymers an exchange of

structurally essential divalent cations in the bacterial membrane leading to the disturbance of

the permeability barrier has been suggested [88,97,161−165]. The high cationic charge

density of peptide-loaded HypoGel resin beads might have the same effect.

3.2.3.3 Effect of particle size

To investigate the effect of the surface area of the peptide-coated solid matrix upon

biological activity, the model KLAL peptide was immobilized on resin beads of different size:

TentaGel MB NH2 and TentaGel M NH2 (Table 9). The surface area of TentaGel M NH2 is

closer to the size of a bacterium ( 0.5 × 2.0 m; along the short and long axes of the

ellipsoidal body) than that of TentaGel MB NH2. However, both resin beads have an identical

overall loading capacity (0.20.3 mmol/g). Taking the diameters of spherical beads and the

number of beads in 1 g resin (Table 9), the surface area of TentaGel M NH2 is calculated to be

almost 35 times larger than that of TentaGel MB NH2.

The density of C-terminally immobilized KLAL on TentaGel MB NH2 and

TentaGel M NH2 was 0.167 and 0.102 mol/mg, respectively (Table 14). Inverse to the

density, KLAL immobilized on TentaGel M NH2 was more active than the

TentaGel MB NH2-tethered peptide against Gram-positive and Gram-negative bacteria


64

(Table 14). According to the suggested mode of action, the peptide accumulates onto the

membrane of bacteria until it reaches a threshold concentration followed by lysis of the

membrane in a carpet-like mode [44]. The results obtained with tethered KLAL on

microspheres and macrobeads suggest that the small micospheres allow a higher local peptide

concentration on the bacterial membrane due to the decreased constraint induced by the small

size of the resin beads. This leads to higher antimicrobial activities for TentaGel M NH2-

bound KLAL in comparison with the tethered peptide on TentaGel MB NH2 beads. Moreover,

the kinetics of dye release from LUVs show that TentaGel M NH2-bound KLAL at

CKLAL 0.26 mM is more active than TentaGel MB NH2-bound KLAL at CKLAL 0.42 mM

(Fig. 33).

Table 14. Antimicrobial activities against B. subtilis and E. coli bacteria and peptide densities of C terminus tethered model KLAL peptide on TentaGel M NH2 (microsphere) and TentaGel MB NH2 (macrobead).

MIC

Bead B. subtilis (DSM 347) E. coli (DH 5)

Density of peptide

(mol/mg) Bead

(mg/ml) Immobilized

peptides (mM)

Bead (mg/ml)

Immobilized peptides

(mM) TentaGel M NH2 0.102 2 0.20 25 2.55

TentaGel MB NH2 0.167 6 1.00 36 6.01

Fig. 33. Kinetics of dye release from POPC/POPG (3/1 [mol/mol]) LUVs induced by TentaGel M NH2- and TentaGel MB NH2-tethered KLAL. Dye release was monitored as increase in the fluorescence intensity at

514 nm at cL 25 M. The concentrations of immobilized KLAL on (♦) TentaGel M NH2 and (◊) TentaGel MB NH2 are 0.26 mM and 0.42 mM, respectively.


65

Taken together, these results suggest that enlargement of the surface area available for

peptide tethering may lead to enhanced peptide aggregation onto the bacteria membranes and

thus, enhanced the antibacterial activity of CAPs with a membrane-lytic mode of action.

3.2.4 Peptide-tethering as a strategy to investigate the mode of

action of CAPs

3.2.4.1 Characterization of MEL, BUF, and TP peptides

MEL (26 amino acids) shows antibacterial and hemolytic activity. It has five basic

residues with two -helical segments, which have been connected together with a helix-

breaking P residue. Basic and hydrophobic residues are located mainly at the C termius and

N terminus, respectively (Table 15). The hinge allows the N terminus and C terminus helices

to localize independently upon interaction with the bacteria membrane. The hydrophobic

flexible “GIG” hinge sequence at the N terminus has been proposed to be important for

insertion into the lipid bilayer [10]. Membrane insertion and association of the peptides into

ion-permeable pores, as proven by discrete conductivity levels, has been suggested to lead to

permeabilization of the bacteria cell membrane by toroidal pore formation (see Fig. 7) [166].

BUF (21 amino acids) is selectively active against bacteria. It is highly basic with

charged residues (+7 including one H residue) dispersed throughout the sequence (Table 15).

BUF is not membrane active and the peptide appears to target intracellular nucleic acids after

translocation across lipid bilayers without significant permeabilizing activity [167]. A hinge

induced by a P residue within the sequence was found to be responsible for translocation

across the cell membranes [46].

TP (13 amino acids) is a cathelicidin-derived CAP, which is rich in R, W, and

P residues (Table 15) [168] with membrane permeabilizing activity as well as the ability to

translocate across the cytoplasmic membrane [119]. The peptide has four R residues localized

at the peptide termini with three W residues in the center of the sequence. The presence of

two P residues induces a unique amphipathic -turn structure in interaction with detergent

micelles [64].


66

Table 15. Amino acid sequences, antimicrobial activities, calculated and observed molecular masses, and tR-RP-HPLC of the peptides.

Molecular mass

[M+3H]+3 (Da.)

MIC (M)*

NO Peptide denotation Amino acid sequence Observed Calculated tR (min) B subtilis (DSM 347)

E. coli (DH 5)

1 MEL GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 949.50 949.26 23.2 1.6 12.5

2 MEL-AOA GIGAVLKVLTTGLPALISWIKRKRQQK(AOA)-NH2 1016.26 1016.29 22.5 0.8 12.5

3 AOA-MEL AOA-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 973.86 973.59 23.4 1.6 50.0

4 BUF TRSSRAGLQFPVGRVHRLLRK-NH2 811.74 811.82 12.9 25.0 6.3

5 BUF-AOA TRSSRAGLQFPVGRVHRLLRKK(AOA)-NH2 878.83 878.85 12.8 25.0 25.0

6 AOA-BUF AOA-TRSSRAGLQFPVGRVHRLLRK-NH2 836.45 836.16 12.9 50.0 25.0

7 TP VRRFPWWWPFLRR-NH2 634.70 634.36 21.1 1.6 6.3

8 TP-AOA VRRFPWWWPFLRRK(AOA)-NH2 701.73 701.39 18.0 1.6 6.3

9 AOA-TP AOA-VRRFPWWWPFLRR-NH2 659.03 658.70 17.9 0.8 12.5 *Values represent the means of the results of three independent experiments performed in triplicate, with standard deviations of less than 5% after 17 h of incubation at 37 °C (Fig. 39).


67

The peptide sequences, the AOA-modified analogs and the tR-HPLC values are

presented in Table 15. AOA modification influenced the peptide hydrophobicity. Whereas,

modification of MEL at the C terminus resulted in a more hydrophilic peptide (tR 22.5 min

compared with tR 23.2 min for the parent peptide), no difference in the tR values was seen

between MEL and AOA-MEL (Table 15). In contrast, AOA modification at peptide termini

had no effect upon the tR values for both BUF and TP. However, TP-AOA and AOA-TP were

more hydrophilic than TP (Table 15).

3.2.4.2 Characterization of tethered MEL, BUF, and TP peptides

MEL, BUF, and TP were immobilized at the C terminus and N terminus on

TentaGel S NH2 resin beads using the oxime-forming ligation strategy (Fig. 27). HPLC-

purified AOA-modified peptides were used. Linkage of peptides at the N terminus results in

reduction of one positive charge compared with the C-terminally modified counterparts. The

tethering was confirmed by the immobilization of the peptides on TentaGel S RAM as

described before (Fig. 34). The derived molecular masses of the immobilized MEL, BUF, and

TP correspond to the theoretical values.

The density of MEL and TP tethered at the C terminus and N terminus on

TentaGel S NH2 was almost identical (~ 0.02 mol/mg for MEL, ~ 0.15 mol/mg for TP)

whereas for BUF, the density of tethered peptide at N terminus was higher (Table 16).

3.2.4.3 Biological activities of free and tethered peptides

Antimicrobial activity of free peptides

The peptides showed antimicrobial activities at micromolar concentrations (Table 15).

MEL and TP peptides are more active against B. subtilis than E. coli with the MIC ranging

between 0.81.6 M. Whereas the peptide activity against B. subtilis follows the order

MEL TP BUF, the compounds showed comparable MIC values against E. coli

(between 6.312.5 M). The influence of AOA-modification is low upon anti-B. subtilis

activity. However, a 4fold reduction in the antimicrobial activities of AOA-MEL, BUF-

AOA and AOA-BUF against E. coli compared with the corresponding parent peptides was

found.


68

200 400 600 800 1000 1200 1400 1600m/z

%

0

779.9540

779.6720391.2790

624.1735623.9544

780.1842

1039.5692

1039.2695

781.4322

1039.9379

1040.2810

1078.2970

1078.6549 1559.3278

A 100

200 400 600 800 1000 1200 1400 1600m/z

%

0

317.2473

1034.9291747.9655

747.7040

317.2899

598.7571

748.2126

996.9324

748.7068

1035.2965

1035.5872

1072.90491665.9164

B100

100

200 400 600 800 1000 1200 1400 1600m/z

%

0

720.0113

719.6761

511.7528

512.0175

720.3394

1136.5165

1136.0056720.6677

721.0103

1137.0634

1137.5294

F

100

200 400 600 800 1000 1200 1400 1600m/z

%

0

978.1696

733.8638

705.1141

676.8695

541.5003

734.1303

734.3969

978.4939

978.5437

1016.17891016.5179

1523.77391016.86541524.3032

C

200 400 600 800 1000 1200 1400 1600m/z

%

0

935.4427

935.1174

673.3438

673.3230

391.2672

673.5991

701.8394

935.7924

973.4582

973.77341459.6752

974.18001459.1674

1516.6615

D100

100

200 400 600 800 1000 1200 1400 1600m/z

%

0

762.6996

762.3692

543.7828

543.7703

391.2735

763.0374

763.3678

1200.5790

1200.05371201.0669

E

Fig. 34. LC-MS spectra of tethered (A) MEL-AOA, (B) AOA-MEL, (C) BUF-AOA, (D) AOA-BUF, (E) TP-AOA, and (F) AOA-TP. The ligation products result from the reaction between the AOA-modified

peptides and TentaGel S RAM pretreated with pyruvic acid. (A) [M+2H]2+ = 1559.33, [M+3H+TFA]3+ = 1078.30, [M+3H]3+ = 1039.57, [M+4H]4+ = 779.67, [M+5H]5+ = 623.95.

(B) [M+2H+3TFA]2+ = 1665.92, [M+3H+2TFA]3+ = 1072.90, [M+3H+TFA]3+ = 1034.93, [M+3H]3+ = 996.93, [M+4H]4+ = 747.70, [M+5H]5+ = 598.76. (C) [M+2H+3TFA]2+ = 1523.77, [M+3H+3TFA]3+ = 1016.18, [M+3H+2TFA]3+ = 978.17, [M+4H+2TFA]4+ = 733.86, [M+4H+TFA]4+ = 705.11, [M+4H]4+ = 676.87,

[M+5H]5+ = 541.50. (D) [M+2H+4TFA]2+ = 1516.66, [M+2H+3TFA]2+ = 1459.17, [M+3H+3TFA]3+ = 973.46, [M+3H+2TFA]3+ = 935.12, [M+4H+2TFA]4+ = 701.84, [M+4H+TFA]4+ = 673.32. (E) [M+2H+2TFA]2+ = 1200.05, [M+3H+TFA]3+ = 762.37, [M+4H]4+ = 543.77. (F) [M+2H+2TFA]2+ = 1136.01, [M+3H+TFA]3+ = 719.68, [M+4H]4+ = 511.75.


69

Inner and outer membrane-permeabilizing activities of MEL, BUF, and TP peptides

To further analyze the membrane activity of MEL, BUF, TP and their AOA-modified

analogs, we examined the effect of the peptides on the integrity of the inner and outer

membrane of E. coli (strain ML-35p). The results are summarized in Fig. 35. Membrane-

active KLAL peptide was used as the positive control. The peptide concentration was around

the MIC values for E. coli (strain DH 5).

Fig. 35. Kinetics of permeabilization of inner and outer membranes of E. coli (strain ML-35p) induced by MEL, BUF, TP and their AOA-modified analogs. The inner and outer membrane permeabilizing activities

result from the cleavage of ONPG and NCF, respectively. The panels indicate (A) intact, (B) C terminus AOA-modified, and (C) N terminus AOA-modified peptides. The peptide concentration was in the range of the MICs

values for E. coli (DH 5) growth.

KLAL and the MEL peptides permeabilize the outer membrane of E. coli in a few

minutes at concentrations of 3 M and 10 M, respectively. This is followed by a fast

permeabilization of the inner membrane confirming the high membrane-lytic activity of the

peptides. Compared with MEL, the TP peptides could permeabilize the membranes at a


70

concentration of 9 M; however, the kinetics of inner membrane permeabilization are slow

(time period of ~ 1 h).

BUF and its AOA-modified analogs do not show significant permeabilizing activities

at concentrations of 10 M and 30 M respectively, the activity patterns were comparable to

the buffer control. The good antimicrobial activity (Table 15), but inability to permeabilize

the bacterial membranes, is consistant with a membrane-independent mechanism of action as

previous studies have suggested [46,167,169].

Antimicrobial activity of tethered peptides

Table 16 summarizes the biological activities of MEL, BUF, and TP immobilized on

TentaGel S NH2 against B. subtilis and E. coli. The activity profiles of bound MEL and TP

against B. subtilis and E. coli are comparable to those of the free peptides. Both tethered

peptides were more active against B. subtilis than E. coli. However, MIC values for the C-

terminally tethered MEL against B. subtilis and E. coli (0.06 mM and 0.60 mM, respectively)

are much lower than the MICs of the N-terminally tethered MEL (up to a factor 5). This

position-dependent activity indicates that the N terminus of MEL should be presented for

optimal interaction with bacteria cell membrane and supports the suggested mode of MEL

action: Insertion of the peptide’s hydrophobic N-terminal domain into the membrane while

the charged C terminus is located in the membrane surface. A similar membrane localization

has been suggested for the highly antimicrobial and hemolytic cupiennin 1 peptide [170],

which has a structural similarity to MEL [166]. The polar C terminus modulates the peptide

accumulation at negatively charged cell surfaces via electrostatic interactions [171]. The

hydrophobic N terminus inserts into the hydrophobic core of the lipid membrane and acts as a

driving force for pore formation and membrane disruption. Membrane insertion of both

peptides results in formation of an ion-conducting pathway.

No differences in the activities of TP immobilized at the C terminus or N terminus

exist against B. subtilis (MIC = 0.74 mM) or E. coli (MIC = 2.94 mM). With TP insertion into

the membrane, the three consecutive W residues form a large hydrophobic domain and the

charged termini remain exposed to the surface [120]. Lipid displacement and pore formation

are the consequences. Tethering of the peptide at both termini has obviously little influence

upon membrane localization and organization, thus excluding a mechanism, which is related

to deep penetration or translocation across the membrane.


71

Table 16. The antimicrobial activities of MEL, and BUF tethered on TentaGel S NH2 beads against B. subtilis and E. coli and densities of the tethered peptides.

MIC

B. subtilis (DSM 347) E. coli (DH 5) Peptide denotation

Position of immobilization

Density of peptide

(mol/mg) Bead

(mg/ml) Immobilized

peptides (mM)

Bead (mg/ml)

Immobilized peptides

(mM) MEL C terminus 0.020 3 0.06 30 0.60

N terminus 0.022 15 0.33 60 1.32

BUF C terminus 0.067 ND ND 28 1.88

N terminus 0.099 ND ND 24 2.38

TP C terminus 0.147 5 0.74 20 2.94

N terminus 0.147* 5 0.74 20 2.94 *Because of the absence of any free amino group in the case of the N terminus tethered TP, the density of the peptide was taken as identical as the value for the C terminus tethered TP for the data analysis.

So far, no convincing results were obtained for immobilized BUF (Table 16). Because

BUF has interacellular targets [46,167,169], it has to translocate across the bacterial

membrane. It is expected that immobilization excludes the peptide from the cytoplasm and

thus abolishes the biological effect (studies will be continued). For nisin, Joshi et al. showed

that the covalent immobilization results in the loss of antimicrobial activity [105]. Nisin

belongs to the group of lantibiotics [172] and targets the lipid II, which is located on the

cytoplasmic side. The authors immobilized thiolated nisin on poly[ethylene oxide]-

poly[propylene oxide]-poly[ethylene oxide] triblocks via formation of a disulfide bridge, but

the peptide was active against bacteria only after reduction of the disulfide bridge, which

means after peptide release. The peptide had to be free for migration of its C terminus across

the cell membrane and the formation of nisin-lipid II complexes [173].

3.2.4.4 Bilayer permeabilizing activities of free and tethered MEL, BUF,

and TP peptides

The bilayer-permeabilizing activities of the peptides against negatively charged

POPC/POPG (3/1 [mol/mol]) LUVs mimicking the charge properties of the lipid matrix of

the inner membrane of Gram-negative bacteria (Fig. 36) correlate well with the activity

profile derived for peptide-induced inner membrane permeabilization (Fig. 35). The activity

of the peptides decreases according to the following order:

MEL peptides > TP peptides >> BUF peptides. As MEL, TP and BUF showed comparable


72

antimicrobial activity against E. coli (Table 15), the results suggest different bactericidal

modes of action for the peptides. Unlike the channel-forming MEL, Yang et al. suggested that

the effect of TP is due to depolarization of the bacterial cell membrane, coupled to targeting

intracellular components [119]. In contrast, the BUF peptides showed no permeabilizing

activity even at the concentration of 400 M, which is consistent with the results of

Kobayashi et al. [167,169] and the idea of the peptide translocation across the cytoplasmic

membrane to reach intracellular targets.

Fig. 36. The bilayer permeabilizing activity of MEL, BUF, TP and their C terminus and N terminus AOA-modified analogs. EC25 was determined at cL 25 M in buffer after 1 min.

MEL, TP and BUF immobilized at the C terminus and N terminus confirmed the

activity pattern (Fig. 37). BUF and TP are both inactive and the kinetics of peptide induced

bilayer permeabilization is slow at about 1 mM concentration. In contrast to the free peptides,

tethered BUF peptides cause higher fluorescence intensity compared to the tethered TP

peptides. Considering the density of loaded peptides (Table 16), this might be due to the light

scattering induced by higher amount of resin used for BUF peptides (Fig. 37). On the other

hand, C-terminally tethered MEL induced dye release more efficiently than the peptide bound

at the N terminus at much lower concentration (0.1 mM). The dependency of activity upon the

site of MEL immobilization again underlines the role of the hydrophobic N terminus domain

for membrane insertion.


73

Fig. 37. Kinetics of dye release from POPC/POPG (3/1 [mol/mol]) LUVs induced by TentaGel-tethered MEL, BUF, and TP immobilized at C terminus and N terminus. Dye release was monitored as an increase in the fluorescence intensity at 514 nm at cL 25 M. The concentration of C-terminally immobilized peptides are

(●) MEL: 0.10 mM, (■) BUF: 1.34 mM, and (▲) TP: 1.47 mM, and for those of N-terminally immobilized

peptides are (○) MEL: 0.11 mM, (□) BUF: 1.98 mM, and ( ) TP: 1.47 mM.

In summary, the distribution of the hydrophobic and charged amino acid residues

within a peptide sequence is an important issue, which has to be taken into account for

preparation of tethered peptides. Because the free hydrophobic region within a peptide

sequence has to insert into the cell membranes in order to disturb their integrity, this domain

should always be far away from the site of immobilization. Whereas, for peptides, such as

KLAL and MK5E with almost identical distribution of positively charged amino acid residues

along the sequenece, and TP with its charges at the sequence termini, the activities of the

immobilized sequences are not position dependent (Tables 12 and 16), MEL should be tetherd

specifically at C terminus for maximal activity. Moreover, not all CAPs may be active against

bacteria when they are tethered. The primary support comes from the resin bead-tethered

BUF, which causes neither cell death nor permeabilization of lipid bilayers.

3.2.5 Summary

Contact-active cationic antimicrobial surfaces have been proposed to exert their

bactericidal effect by penetrating the bacterial cell wall if their biologically active site is far

enough away from the surface of the solid matrix (Fig. 38). Autolysis, initiated by the

exchange of membrane stabilizing cations, has been suggested as the mode of action

[88,97,161−165]. Because of the toxicity of these surfaces to human cells, in recent years


74

increasing attention has been focused upon surfaces covered with CAPs. Among the various

methods of immobilization of CAPs, covalent attachment offers several advantages such as

long-term stability and lower toxicity of the biomolecules compared to their incorporation

into release-based systems [101]. However, the application of the appropriate chemistry for

peptide tethering on materials, which induces little modification of the peptide structural

parameters, such as conformation, charge, hydrophobicity and amphipathicity is of particular

importance [16].

Fig. 38. Scheme of antimicrobial action of immobilized biocides via a polymeric spacer.

Chemical strategies including SPPS, thioalkylation and oxime-forming ligation were

used in order to link CAPs with different activity spectra and modes of action at the

C terminus, N terminus, and side chains on PEGylated resin beads as model solid surfaces

(Fig. 27). The CAPs include membrane-active peptides, such as model KLAL peptide [44],

MG 2-derived MK5E [118], MEL [8], BUF [46,167,169], and TP [119,120,168].

Reasonable antimicrobial activity of surface-bound peptides requires the optimization

of the coupling parameters such as the length of the spacer, the surface area of solid matrices,

and the amount of target-accessible peptide. Studies with the tethered membrane active

peptides, e.g., KLAL, and MK5E, on TentaGel S NH2 resin (the resin, which is characterized

with the PEG spacers (3 kDa) (Table 9) long enough to span the thickness of bacteria cell


75

membrane) demonstrated that tethering conserved the activity spectrum of the peptides at

reduced concentrations (Table 12). The antimicrobial activity distinctly decreased with

reduction of the spacer length and even an increase in the loading capacity of the resin, e.g.,

HypoGel 200 NH2, was not sufficient to compensate for the spacer length-related activity

decrease (Tables 12,16). Furthermore, an increase in surface area of the solid matrices for

peptide tethering improved the antimicrobial activity. The analyzed parameters are relevant

for the establishment of a more general approach to obtain efficient biocidal solid matrices

loaded with CAPs.

Furthermore, the studies demonstrate that immobilization can be used as a powerful

tool to gain insight into the modes of peptide interaction with biological and model

membranes. Differences in the activity of N- and C-terminally tethered MEL confirmed the

important role of the MEL N terminus for insertion and peptide orientation within the lipid

matrix to form pores, whereas the independency of KLAL activity upon the site of

immobilization confirmed an orientation of the helix parallel to the lipid bilayer and a carpet-

like mode of action. Hopefully, ongoing studies on immobilized BUF would enable us to

distinguish between the peptides with a membrane-perturbing mode of action and those with

intracellular targets.

Summary

76

4 Summary CAPs are components of the innate immune system of mammalians and play an

important role in the defense of all organisms, including plants, against invading pathogens.

Their mode of action, based on interaction with the cell membrane and their properties, such

as rapid action and a low tendency to stimulate the bacterial resistance have been considered

as a promising basis for the development of a new class of antibiotics, particularly for topical

application. In recent years, much effort has been focused upon modifying such peptides with

respect to antimicrobial activity. However, only identification of the structural motifs and

optimization of peptide interactions with the different classes of pathogens, such as Gram-

positive or Gram-negative bacteria will provide the basis for the development of highly

selective antimicrobial compounds. Furthermore, increasing efforts have been focused upon

the development of antimicrobial surfaces, which inhibit the growth of bacterial adhesion and

subsequent biofilm formation.

4.1 W-substituted c-WFW analogs

In the first part of this work, it was planned to identify interaction moieties on the

cellular level and the driving forces responsible for the pronounced activity increase in RW-

rich hexapeptides after induction of conformational constraints by peptide cyclization, using

systematic modification of the hydrophobic cluster. For this, a small library of peptides based

upon the sequence of cyclo-RRRWFW was prepared, in which W residues were replaced by

unnatural analogs such as Dht, Igl, 5MeoW, 5fW, 5MeW, 1MeW, Bal and the -amino acid

b3-hW with altered hydrophobicity, dipole and quadrupole moments, ability of hydrogen

bonding, amphipathicity and flexibility of the cyclic peptide. This made allowed for the

undertaking of a systematic investigation of aromatic clusters that affect peptide interactions

with lipid systems mimicking the outer and inner membranes, as well as of the biological

activities of peptides against E. coli, in comparison with Gram-positive B. subtilis and red

blood cells.

To understand the effect of the peptides upon both bacteria and eukaryotic cell

membranes in detail, the peptides’ interactions with lipid bilayers, i.e., POPC, POPC/POPG

(3/1 [mol/mol]) as models of the target membrane and POPC/LA (12/1 [mol/mol]), POPC/r-

LPS (12/1 [mol/mol]), and POPC/s-LPS (12/1 [mol/mol]), as models of the outer membrane

of Gram-negative bacteria were studied by isothermal titration calorimetry. Thermodynamic

Summary

77

parameters for peptide-lipid interaction were determined using a surface partitioning

equilibrium model and correcting for electrostatic effects by the Gouy-Chapman theory and

the results were correlated with the biological data.

The study showed that peptide activity against erythrocytes and bacteria can be

explained on the basis of peptide accumulation at the lipid matrices of the target membranes

driven by electrostatic interactions and subsequent membrane partitioning determined by

hydrophobic interactions. Peptide hydrophobicity and backbone constraints are the crucial

determinants of biological activity. Other modifications in the hydrophobic cluster of the

cyclic hexapeptides have minor influence upon peptide interaction with biological systems

and model membranes.

The different susceptibilities of E. coli and B. subtilis can be explained by differences

in the negative surface charge of the plasma membranes. Strong peptide interactions with the

outer-membrane LPSs of E. coli probably influence the peptide transport across the outer

wall, and thus are responsible for high activities of cycles. The activity of the peptides against

B. subtilis increased with enhanced hydrophobicity. In contrast, any alterations in

hydrophobicity, amphipathicity of the indole ring, and backbone flexibility modulated the

antimicrobial activity against E. coli in a more complex way.

The accumulation of the individual peptides to different lipid systems was found to be

determined by electrostatic and hydrophobic interactions and followed the order: POPC/s-

LPS POPC/r-LPS POPC/POPG = POPC/LA POPC. The hydrophobic contribution to

binding to POPC and mixed POPC/POPG bilayers was comparable. Low hydrophobicity and

conformational flexibility of the peptides reduced partitioning into the layers. The peptide

binding was largely enthalpy-driven, which is in agreement with the nonclassical hydrophobic

effect.

In the presence of r-LPS and s-LPS the modulating role of hydrophobicity in

partitioning of the different cyclic peptides decreased while no influence was found upon the

low affinity of the highly flexible linear parent peptide. For lipid systems with incorporated r-

LPS or s-LPS, the different cyclic peptides showed comparable binding affinities. However,

the K0 values for interaction with POPC/s-LPS systems were almost by one order of

magnitude larger than for binding to POPC/r-LPS bilayers which uncovers the significant role

Summary

78

of the O-antigen and outer core oligosaccharides of LPS for more specific interactions of the

cylic peptides.

The reason why distinct differences in hydrogen bonding ability, dipole moment, and

aromaticity of the W residues are not reflected in the thermodynamic characteristics of the

peptide interactions with LPS-containing lipid bilayers remains to be elucidated.

4.2 Site-specific immobilization of CAPs

In the second part of this work, the influence of immobilization upon the activity

profile of CAPs was analyzed. This study was to analyze a number of coupling parameters

with respect to the use of peptides in the generation of biocidal surfaces. Resin beads bearing

PEG spacers of different lengths and various size distributions (between 10−300 m) were

covered by linkage of an amphipathic model KLAL peptide, a magainin 2-amide-derived

MK5E, melittin, buforin 2, and the RW-rich tritrpticin. The peptides were characterized by

different modes of action. Standard SPPS, thioalkylation and oxime-forming ligation

strategies were used to immobilize the peptides at C terminus and N terminus and via

different side chain positions. The influence of resin bead parameters, such as spacer length,

spacer density, and surface area available for peptide attachment were studied by the

investigation of antimicrobial and bilayer permeabilizng activities of the peptides covered

surfaces. Additionally, covalent immobilization of the CAPs to an insoluble solid material in

combination with activity studies was developed as an alternative approach to elucidate the

mode of action of the peptides.

The antimicrobial peptides KLAL and MK5E were suitable for the production of

antibacterial surfaces. The tethered peptides also act via permeabilization of the cell

membrane. The free peptides showed antimicrobial activities against B. subtilis and E. coli at

micromolar concentrations. Immobilization on resins reduced the antimicrobial activity

spectra of the free peptides to millimolar concentrations. The activity profile against Gram

negative and Gram positive bacteria and red blood cells remained constant.

The length of spacer between the solid surface and active sequences is a critical

parameter for peptide activity. The peptide activities decrease with reduction of the spacer

length independent of amount of loaded peptide on solid surface. Furthermore, enhancement

Summary

79

of surface area available for peptide attachment increases the biocidal activity of tethered

peptides.

Depending upon the mechanism of action of peptides, the coupling position affects the

peptide activity. An appropriate coupling position has to be selected depending upon the

orientation of the peptides in the membrane. Supports come from the biological and lipid

bilayers permeabilizing activities of tethered KLAL with a “carpet-like” mode of action as

compared with MEL, which forms pores through the peptide N-terminal insertion and

association. Whereas the activities of tethered KLAL is independent of coupling position, N-

terminal immobilization of MEL leads to the loss of the activities compared with the C-

terminally tethered peptide.

Based upon the relation between peptide activity, their positioning and association

within the membrane, peptide tethering should make it possible to gain insight into the

mechanisms of peptides action according to the “carpet-like mode” or pore formation via the

“barrel stave” or “toroidal model”.

Zusammenfassung

80

5 Zusammenfassung Kationische antimikrobielle Peptide sind Bestandteil des angeborenen Immunsystems

von Säugetieren und spielen eine wichtige Rolle bei der Verteidigung aller Organismen gegen

Krankheitserreger. Ihre Wirkungsweise über die Permeabilisierung der Zellmembran und ihre

rasche Wirkung bei nur geringer Stimulation einer bakteriellen Resistenzentwicklung bilden

eine vielversprechende Grundlage für die Entwicklung peptidischer Antibiotika, insbesondere

für topische Anwendungen. In den letzten Jahren wurde vielfach versucht, Peptide im

Hinblick auf ihre antimikrobielle Aktivität zu optimieren. Um selektive antimikrobielle

Verbindungen zu entwickeln, müssen die Strukturmotive identifiziert werden, die für die

Wechselwirkung der Peptide mit verschiedenen Klassen von Pathogenen, wie z. B. Gram-

positiven oder Gram-negativen Bakterien, entscheidend sind. Darüber hinaus werden große

Anstrengungen unternommen, um antimikrobielle Oberflächen zu entwickeln, die die

bakterielle Adhäsion an Oberflächen und die dadurch ermöglichte Biofilmbildung hemmen

sollen.

5.1 W-substituierten c-WFW Analoga

Im ersten Teil dieser Arbeit sollten sowohl die Interaktionsgruppen auf der Ebene von

Zellmembranen als auch die Triebkräfte identifiziert werden, die für den ausgeprägten

Aktivitätsanstieg bei Zyklisierung RW-reicher Hexapeptide verantwortlich sind. Für die

systematische Modifizierung des hydrophoben Clusters der Peptide, wurde eine kleine

Bibliothek von cyclo-RRRWFW-abgeleiteten Sequenzen synthetisiert, in denen W-Reste

durch unnatürliche Analoga wie Dht, Igl, 5MeoW, 5fW, 5MeW, 1MeW, Bal und die -

Aminosäure, b3-hW ersetzt wurden, was in einer Änderung der physiko-chemischen

Eigenschaften, wie z. B. Hydrophobizität, Dipol- und Quadrupol-Moment, Fähigkeit zur

Wasserstoffbrückenbildung, Amphipathie, und Flexibilität resultierte. Dies machte

systematische Untersuchungen der aromatischen Clustern in Wechselwirkungen mit

Membran-modellierenden Lipidsystemen möglich und erlaubte einen Vergleich der

biologischen Aktivität der Peptide gegenüber Gram-negativen E. coli, Gram-positiven

B. subtilis und roten Blutzellen.

Um die Wirkung der Peptide auf bakterielle und eukaryotische Zellmembranen im

Detail zu verstehen, wurde die Bindung der Peptide an verschiedene Lipid-Doppelschichten,

nämlich POPC und POPC/POPG (3/1 [mol/mol]) als Modelle biologischer Targetmembranen

Zusammenfassung

81

und POPC/LA (12/1 [mol/mol]), POPC/r-LPS (12/1 [mol/mol]) und POPC/s-LPS

(12/1 [mol/mol]) als Modelle der äußeren Membran von Gram-negativen Bakterien, durch

isothermale Titrationskalorimetrie untersucht. Thermodynamische Parameter der Lipid-

Peptid-Interaktionen wurden mittels eines Verteilungs-Modells bestimmt und bezüglich

elektrostatischer Effekte nach der Gouy-Chapman Theorie korrigiert. Diese Ergebnisse

wurden mit den biologischen Daten korreliert.

Die Ergebnisse zeigten, dass die Peptidaktivität gegenüber Erythrocyten und Bakterien

durch die Peptidakkumulation, die durch elektrostatische Wechselwirkungen bestimmt ist

sowie nachfolgende Verteilung in das hydrophobe Innere der Lipidmatrix erklärt werden

kann. Die Peptidhydrophobizität und konformationelle Beschränkungen im Ring sind

entscheidende Faktoren für die biologische Aktivität. Andere Modifikationen in den

hydrophoben Clustern der Hexapeptide haben nur geringen Einfluß auf die Peptidwirkung

gegenüber Zellen und auf Modelmembranen.

Die unterschiedliche Empfindlichkeit von E. coli und B. subtilis Bakterien lässt sich

auf Grundlage der unterschiedlichen Ladung der Plasmamembranen verstehen. Die Aktivität

der Peptide gegen B. subtilis stieg mit erhöhter Hydrophobizität. Im Gegensatz dazu

beeinflußt jede Änderung der Hydrophobizität, Amphipathie des Indolring und Flexibilität im

Rückgrat die antimikrobielle Aktivität gegen E. coli in einer komplexeren Art und Weise.

Starke Interaktionen der Peptide mit dem O-Antigen und Resten der äußeren Region von LPS

in der äußeren Membran Gram-negativer E. coli sind wahrscheinlich für den effizienten

Peptidtransport durch die äußere Barriere und damit die hohe Aktivität der Zyklen

verantwortich.

Die Akkumulation der Peptide an unterschiedliche Lipid-Bilayern wird durch

elektrostatische und hydrophobe Wechselwirkungen bestimmt und folgt der Reihenfolge:

POPC/s-LPS POPC/r-LPS POPC/POPG = POPC/LA POPC. Der hydrophobe Beitrag

zur Bindung an POPC und gemischte POPC/POPG Doppelschichten war vergleichbar.

Geringe Hydrophobizität und konformationelle Flexibilität der Peptide reduziert die

Verteilung in die Lipidschichten. Die Peptidbindung ist weitgehend Enthalpie-getrieben was

dem nichtklassischen hydrophoben Effekt entspricht.

In Anwesenheit von LA, r-LPS und s-LPS sinkt die modulierende Rolle der

Hydrophobizität bei der Verteilung der verschiedenen zyklischen Peptide, wobei kein Einfluß

Zusammenfassung

82

auf die geringe Affinität des hochflexiblen linearen Ausgangspeptids gefunden wurde. Für

Lipid-Systeme mit r-LPS oder s-LPS, zeigten die verschiedenen zyklischen Peptide

vergleichbare Bindungsaffinitäten. Allerdings waren die hydrophoben

Verteilungskoeffizienten für die Interaktion mit POPC/s-LPS-Systeme fast eine

Größenordnung höher als für die Bindung an POPC/r-LPS-Doppelschichten. Das unterstreicht

die bedeutende Rolle des O-Antigens und der Oligosaccharide im äußeren Kern des LPS als

spezifischer Wechselwirkungspartner der zyklischen Peptide.

Der Grund dafür, dass sich deutliche Unterschiede der Peptide in der Fähigkeit,

Wasserstoffbrückenbindungen zu bilden, im Dipol-Moment und in der Aromatizität nicht in

den thermodynamischen Eigenschaften ihren Interaktionen mit LPS-enthaltende Lipid-

Doppelschichten widerspiegeln, bleibt noch aufgeklärt werden.

5.2 Ortsspezifische Immobilisierung von CAPs

Im zweiten Teil dieser Arbeit wurde der Einfluss einer Immobilisierung kationischer

antimikrobieller Peptide auf ihr Wirkprofil analysiert. Mit dieser Arbeit sollte ein Beitrag

geleistet werden, um Peptide für die Entwicklung antimikrobieller Materialien einsetzen zu

können. Dazu wurden Harze verschiedener Korngrößenverteilungen (zwischen 10−300 mm)

und mit PEG-Spacern unterschiedlicher Längen mit dem linearen amphipatischen

Modellpeptide KLAL, einem von Magainin 2-amide abgeleitetem MK5E-Peptid sowie den

Peptiden Melittin, Buforin 2 und dem W-reichen Tritrptizin beladen. Die Peptide zeichnen

sich durch unterschiedliche Wirkungsmechanismen aus. Die Peptide wurden mit Hilfe

verschiedener Synthesestrategien: Standard Festphasen-Peptidsynthese, Thioalkylierung und

Oxim-forming Ligation, über ihren C-terminus, N-terminus oder verschiedene

Seitenkettenpositionen immobilisiert. Der Einfluss der Harzparameter, wie Länge und Dichte

der Spacer oder der für die Peptidanbindung zur Verfügung stehenden Oberfläche sowie die

Rolle der Peptidposition zur Immobilisierung wurde analysiert durch eine Bestimmung der

antimikrobiellen und membranpermeabilisierenden Aktivität der beladenen Harze. Darüber

hinaus wurde die Peptidimmobiliseirung an Syntheseharz als ein alternativer Ansatz zur

Untersuchung der Wirkungsweise der Peptide eingesetzt.

Die antimikrobiellen Peptide KLAL und MK5E sind zur Herstellung antibakterieller

Oberflächen geeignet. Sie wirken auch im immobilisierten Zustand durch Permeabilisierung

der Zellmembran. Die untersuchten freien Peptide zeigten antimikrobielle Aktivitäten gegen

Zusammenfassung

83

B. subtilis und E. coli im mikromolaren Konzentrationen. Immobilisierung am Harz reduziert

die Peptidaktivität auf millimolare Konzentrationen. Das Aktivitätsprofil gegenüber Gram

negativen, Gram positiven Bakterien und roten Blutzellen bleibt jedoch erhalten.

Die Abstand zwischen der festen Oberfläche und der aktiven Sequenzen ist ein

kritischer Parameter für die Peptid-Aktivität. Die Peptidwirksamkeit verringert sich mit

Abnahme der Spacerlänge, unabhängig von der Menge der Peptids auf der Oberfläche.

Andererseits, führt die Vergrößerung der verfügbaren Fläche für die Peptidkopplung zur einer

Verbessrung der bioziden Wrikung.

In Abhängigkeit vom Wirkungsmechanismus, beeinflusst die Kopplungsposition im

Peptid die Aktivität entscheidend. Eine geeignete Kopplungsposition ist in Abhängigkeit von

der Orientierung der Peptide in der Membran zu wählen. So ist die Aktivität von KLAL, für

das eine Lokalisation in der Membranoberfläche und ein “Carpet-like” Wirkungsmodus

vorgeschagen wird, im immobilisierten Zusand weitgehend unabhängig von der

Kopplungsposition. Für Mellitin, das in Lipidschichten Poren bildet durch Insertion des N-

terminus und Assoziation, muß N-terminale Immobilisierung zum Aktivitätsverlust führen.

Dieser Zusammenhang sollte es möglich machen Peptidimmobilisierung als Methode

zu entwickeln, um Aussagen zu den Modellen der Peptidwirkung zu gewinnen, die durch

unterschiedliche Positionierung und Assoziation der Peptide in der Membrane charakterisiert

sind.

Experimental section

84

6 Experimental section

6.1 Materials: Chemicals and reagents

TentaGel S NH2, TentaGel M NH2, TentaGel MB NH2, TentaGel S RAM,

HypoGel 400 NH2, HypoGel 200 NH2, and ClTrt-Cl resins were purchased from

Rapp Polymere GmbH, Germany. The Fmoc-N-protected amino acids were obtained from GL

Biochem (Shanghai) LtdS, China. The side chain protecting groups were as follows:

Arg (Pbf), Asn (Trt), Asp (t-Bu), Cys (Trt), Gln (Trt), Glu (t-Bu), Gly (Boc), His (Trt),

Lys (Boc), Pro (Boc), Ser (t-Bu), Thr (t-Bu), Trp (Boc), and Tyr (t-Bu). We used Fmoc-

Dht(Boc)-OH and Fmoc-Igl-OH (both Advanced ChemTech/ThuraMed, U.S.A.), Fmoc-Bal-

OH and N--Fmoc-b3-hW-OH (both Sigma-Aldrich GmbH, Germany), Fmoc-5Metrp-OH

and Fmoc-5Meotrp-OH (both AnaSpec Inc., U.S.A.), Fmoc-1Metrp-OH (BACHEM,

Switzerland), Fmoc-5Ftrp-OH (Iris Biotech GmbH, Germany), Fmoc-Lys(Dde)-OH and

Fmoc-PEG 2-OH [Fmoc-NH-PEG-COOH (9 atoms)] (both Novabiochem, Germany), Boc-

AOA-OH and pyruvic acid (both Fluka, Germany) from the noted provider. Fmoc-Cl, and

activating reagents, such as HOBt and HBTU were obtained from Iris Biotech GmbH.

Thioanisole, EDT, TIPS, GnHCl, and calcein were provided from Fluka, NCF from Oxoid

(U.K.), and phenol from Honeywell Riedel-de Haen, Germany. 1-PrOH, TBP, ampicillin, and

ONPG were from Sigma-Aldrich GmbH. The culture tubes (reagent and centrifuge tube),

5 mL; 75 12 mm; PS were from SARSTEDT AG & Co, Germany. LB, LB-agar, s-LPS

(source: E. coli K-235), Rd-LPS (source: E. coli F583 (r-LPS)), and diphosphoryl LA (source:

E. coli F583 (Rd mutant)) were obtained from Sigma-Aldrich GmbH. The lipids POPC and

POPG were purchased from Avanti Polar Lipids, U.S.A. TRIS, and other chemicals were

from Fluka, Acros Organics (Belgium), or Merck (Germany).

6.2 Methods

6.2.1 Synthesis of peptides

6.2.1.1 Synthesis of linear peptides (automated synthesis)

The linear peptides were synthesized automatically by SPPS using a standard Fmoc/t-

Bu protocol [149]. Syntheses were carried out on TentaGel S RAM resin (0.26 mmol/g) using

5 equiv. Fmoc-protected amino acids and 4.9 equiv. HBTU as a coupling reagent in the


85

presence of 10 equiv. DIEA in DMF. Double couplings for 20 min were allowed. N terminus

deblocking was carried out twice with 20% piperidine in DMF for 5 min. Washes were made

with DMF. Acetylation was carried out twice and for 10 min using

Ac2O/DIEA/DMF (0.5/1/3.5 v/v/v). For acetylation of the PEGylated peptides the same

methodology was used. The strategy of SPPS was also used to introduce PEG 2. For side

chain PEGylation of K residues, Fmoc-Lys(Dde)-OH was used instead of Fmoc-Lys(Boc)-

OH. The Dde side chain was removed by exposure to 2% hydrazine in DMF twice for 5 and

10 min. After washing, PEG 2 was coupled to the free amino group using HBTU activation.

Final cleavage from the resin and deprotection of the side chain functionalities were achieved

by exposure to a mixture of 5% phenol, 2% TIPS, and 5% water in TFA for 2 h. After

cleavage of the protecting groups, peptides were precipitated by addition of cold diethyl ether.

6.2.1.2 Synthesis of cyclic peptides (manuel synthesis)

The peptide acids, as precursors for the preparation of cyclic peptides, were

synthesized manually by SPPS using the standard Fmoc/t-Bu protocol. Syntheses were carried

out on ClTrt-Cl resin (1.03 mmol/g) using Fmoc-protected amino acids. Before the synthesis,

the resin was placed in a reaction vessel equipped with a porous frit and swollen in DCM for

30 min followed by filtration of the solvent under vacuum. The peptides were synthesized

according to the sequence RRRXFX (X = W and other unnatural amino acids). To couple the

first amino acid, i.e, Fmoc-Trp(Boc)-OH or the unnatural amino acids, 1 equiv. of the

protected amino acid and 1 equiv. of DIEA were dissolved in DCM and added to the resin.

The mixture was agitated on a shaker for 2 h. Afterwards, the vessel was drained and

subjected to a mixture of DCM, methanol, and DIEA (8/2/1 [v/v/v]) twice for 10 min to block

any unreacted chloro- functional groups on resin. The resin was washed again with DCM five

times for 1 min and drained under vacuum. The final loading of the coupled amino acid was

quantified by UV absorption of the released Fmoc chromophor at 301 nm. The resin was then

washed with 20% piperidine in DMF twice for 5 min to deblock the Fmoc group and washed

again with DMF five times. The synthesis was continued to the end by utilizing the

HBTU activation, i.e., 3 equiv. amino acids, 3 equiv. HBTU and 6 equiv. DIEA in respect to

the capacity of the resin in DMF. Double couplings for 20 min were allowed and washes were

conducted with DMF. Final cleavage from the resin and deprotection of the side chain

functionalities were achieved by exposure to a mixture of 5% phenol in TFA for 2 h. In the

case of sequences with W-analogs, in which the indole moiety is unprotected, the


86

reagent K (TFA/thioanisole/water/phenol/EDT [82.5/5/5/5/2.5 v/v]) was applied as the

cleavage cocktail. After cleavage, the peptides were precipitated by addition of cold diethyl

ether. The crude peptides were dissolved in a mixture of water/ACN (1/1 [v/v]) and the

resulting solution was lyophilized. The head-to-tail cyclization of the crude linear peptides

was performed by the use of HAPyU and DIEA in DMF under dilute condition as described

before [53].

6.2.1.3 HPLC purification of crude peptides

The crude peptides were dissolved in a mixture of water/ACN (1/1 v/v) and purified

by RP-HPLC on a Shimadzu LC-10A system (Japan) using a PolyEncap C-18 column

(250 × 20 mm, 10.0 m, 300 Å) (Bischoff Analysentechnik, Germany) operating at 220 nm

and 10 mL / min to produce final products that were more than 95% pure. The sample

concentration was 5 mg of peptide / mL in water/ACN (1/1 v/v). The mobile phase A was

0.1% TFA in water, and phase B was 0.1% TFA in 80% ACN−20% water (v/v) using a linear

gradient 5 to 95% B in 40 min. The compounds were further characterized either by ESI-MS

in the positive ionization mode using an ACQUITY UPLC system with LCT Premier

mass spectrometer (Waters Corporation, U.S.A.) or by MALDI-MS (MADLI II, U.K.) to

confirm the right molecular mass.

6.2.1.4 Characterization of peptides based upon tR-HPLC.

Measurement of the tR value of CAPs using RP-HPLC is a convenient method to

compare peptide hydrophobicity. This method is based on the ability of the hydrophobic

residues in a peptide to interact with the hydrophobic surface of the HPLC reversed stationary

phase. Thus, the differences due to increases in hydrophobicity are reflected in the retention

time tR.

Chromatographic characterization was performed on a Jasco analytical HPLC system

(Jasco, Japan) with a diode array detector operating at 220 nm. Runs were carried out using a

PolyEncap C-18 column (250 × 4.0 mm, 5.0 m, 300 Å) (Bischoff Analysentechnik)

operating at 220 nm and 1 mL / min. The sample concentration was 1 mg of peptide / mL in

eluent A. The mobile phase A was 0.1% TFA in water, and phase B was 0.1% TFA in

80% ACN−20% water (v/v). The tR of the peptides was determined using a linear gradient of

5 to 95% phase B over 40 min at room temperature.


87

6.2.2 Immobilization of CAPs

6.2.2.1 SPPS: C terminus immobilization

C-terminally immobilized model KLAL peptide and MK5E were prepared by the use

of automated peptide synthesis as described above except that: TentaGel S NH2,

HypoGel 200 NH2, HypoGel 400 NH2, TentaGel MB NH2, and TentaGel M NH2 were used

for the synthesis. The immobilized peptides were deprotected by TFA cleavage as mentioned

above. The resins were then washed several times with diethyl ether, DCM, DMF, 5% DIEA

in DCM, and DCM. Afterwards, the peptide-loaded resins were dried under vacuum and

stored at 4 °C.

6.2.2.2 Thioalkylation: N terminus and side chain immobilization of KLAL

An additional C residue was introduced at the N terminus or an -amino group of the

K residues of the peptides. BrAcOH (Fluka, Germany) was introduced at the amino-

functionalized resin using its anhydride, which was prepared in situ by mixing

BrAcOH (3 equiv.), DIC (3 equiv.) and HOBt (3 equiv.) in DCM for 2 h. After washing

and drying the resins overnight under vacuum, the Cys-modified peptides [~ 15 mM in

DMF/1-PrOH (1/4 v/v), TBP (3 equiv.), DIEA (6 equiv.)] were added to the BrAcOH-

modified resins and incubated by vortexing overnight at room temperature. The peptide-

bearing resins were washed with DMF, followed by DCM and then dried under vacuum and

stored at 4 C (Fig. 27).

6.2.2.3 Oxime-forming ligation: N terminus and side chain immobilization

of MK5E, MEL, BUF, and TP

AOA was introduced at the C terminus, N terminus or an -amino group of K residues.

For the C terminus immobilization of the peptides, we introduced an extra K residue at the

first position of the C terminus of the sequences. Fmoc-Lys(Dde)-OH was used for this

purpose. To overcome the problem of over-acetylation of nitrogen atoms in the

─NH─O─ moiety, which would lead to AOA(AOA)-peptides during the synthesis of an

aminooxy-peptide for chemical ligation [174], the aminooxy-peptides were prepared by using

3 equiv. DIC, 3 equiv. HOBt as coupling reagents and 3 equiv. AOA in DCM for 1 h. The

ketone-containing resin was prepared as followed: Three equiv. of pyruvic acid were added to


88

the reaction vessel containing TentaGel S NH2, 3 equiv. DIC and 3 equiv. HOBt in an

appropriate amount of DCM. After 2 h, the reaction was followed with the Kaiser test.

Finally, the resin was washed several times with DMF and DCM and subsequently dried

under vacuum overnight for further use. Peptides modified with AOA (~ 15 mM in

acetate buffer, 6 M GnHCl, pH 4.6) were added to the dry ketone-functionalized resin and

allowed to react at room temperature overnight. After washing with DMF followed by DCM,

the resins were dried under vacuum and stored at 4 °C (Fig. 27).

6.2.2.4 Characterization of tethered peptides using UV-absorption of the

Fmoc-chromophore

The density of immobilized peptides was determined by measuring the absorption of

the cleaved Fmoc-chromophore upon treatment of the peptide-loaded resins

with 20% piperidine in DMF. The resin-bound peptides were exposed to a 5-fold excess of

Fmoc-Cl, and DIEA in DCM (calculated on the basis resin capacity and the number of

available amino groups in the sequence of the individual peptide). After 1 h, the resin was

washed in DCM and subjected to 20% piperidine in DMF for 20 min. Afterwards an aliquot

of the supernatant was added to a cuvette containing piperidine and the absorption was

measured at 301 nm ( = 6000 M-1

.cm-1

) using a Lambda 9 spectrophotometer (Perking-

Elmer, Germany).

6.2.3 Antimicrobial activity

Antibacterial activities were assessed against Gram-negative E. coli (strain DH 5)

and Gram-positive B. subtilis (strain DSM 347).

6.2.3.1 Bacterial culture preparation and determination of MIC and MBC

Bacteria were grown at 37 °C, shaking at 180 rpm, in LB to the mid-log phase as

determined by the optical density (OD600 of 0.4−0.5). The bioassays with resin-bound and free

peptides were carried out in culture tubes and 96-well microtiter plates respectively (Figs. 39,

40).


89

Fig. 39. Determination of MIC of bacterial growth of free peptides using microtiter plate method. MIC values represent the means of three independent experiments performed in triplicate.

Fig. 40. (A) Determination of MIC of bacterial growth of tethered peptides. (B) Determination of MBC on an LB-agar plate. Each MIC of peptide-covered resin was determined in one experiment using a serial dilution

of the peptide-loaded resin. The values were confirmed in two independent experiments using the determined MIC and two resin concentrations below and above the MIC.

Appropriate amounts of peptide-bearing resin were added to the culture tubes

containing 1 mL of LB medium. An aliquot of the cell suspension was then added resulting in

a cell concentration of ~ 1.6 × 106 cells/mL. Non-modified resins were used as a control.

Depending upon the cell line, the tested concentrations ranged between 0.5 and 80 mg/mL.

For free peptides, 150 L of the bacterial suspension was added to 50 L of the culture


90

medium containing the peptides at various concentrations. The final cell concentration

was ~ 1.6 106 cells/mL. The final concentrations of peptides ranged from 100 to 0.05 M in

2-fold dilutions. Cultures without peptides were used as control. The microtiter plates were

incubated at 37 C with shaking (180 rpm). After 17 h, the absorbance was read at 600 nm

(Safire Microplate Reader, Tecan, Germany). The MIC was determined as the lowest peptide

concentration at which no bacterial growth was observed. To evaluate the antimicrobial

activity of the immobilized peptides, test tubes were shaken horizontally in order to enhance

the probability of cell-resin contact. This procedure provided reproducible values. After 17 h

the MICs of immobilized peptides (mM) were determined on the basis of the determined

resin-related MIC values (mg/mL) taking into consideration the amount of immobilized

peptide (mol) per mg resin. To determine the MBC, an aliquot (200 µL) from the wells with

peptide concentrations ≥ MIC was spread on an LB-agar plate. After incubation at 37 C for

24 h, the number of colonies was counted. The MBC was defined as the lowest peptide

concentration at which no colonies were detected (Fig. 40B). The experiments were

performed in triplicates.

6.2.4 Hemolytic activity

The hemolytic activity of the peptides was determined using human RBCs (Charité–

Universitätsmedizin, Germany). Prior to the assay, the erythrocytes were washed several

times in buffer (10 mM TRIS, 150 mM NaCl, pH 7.4). 100 L cell suspension

(2.5×109 cells/mL), varying amounts of the peptide stock solution (concentration usually 10-3

and 10-4 M in TRIS buffer) and buffer were pipetted into Eppendorf tubes to give a final

volume of 1 mL. For determination of the resin, appropriate amounts of non-modified

TentaGel S NH2 were added to 100 L cell suspensions and 900 L buffer. Afterwards, the

suspensions containing 2.5 × 108 cells/mL were incubated for 30 min, with gentle shaking, in

an Eppendorf thermomixer. After cooling in ice water and centrifugation at 2000 × g and 4 °C

for 5 min, 200 L of the supernatant was mixed with 2300 L of 0.5 % NH4OH, and the

OD540 was determined at 540 nm. Zero hemolysis (blank) and 100% hemolysis (control) were

determined with cell suspensions incubated in buffer or 0.5% NH4OH, respectively.


91

6.2.5 Inner and outer membrane permeabilizing activities of

MEL, BUF and TP and their AOA-modified analogs

Most CAPs act via permeabilization of the bacterial cell membrane. In order to assess

the permeabilizing activity of the peptides in vitro, Lehrer et al. developed a protocol, which

allows the study of the Gram-negative bacterial inner and outer membrane permeabilizing

activity of CAPs [175].

Fig. 41. Schematic representation of the assay used for determination of the E. coli inner and outer membrane permeabilizing activity of CAPs.

To perform this assay, the E. coli (strain ML-35p) was used. The cells are

characterized by a lack of lactose permease. The strain expresses periplasmic -lactamase and

cytoplasmic -galactosidase. Due to the lack of lactose permease, NCF (-lactamase

substrate) and ONPG (-Galactosidase substrate) are blocked from being transported across

the outer and inner membrane of the bacterial cell respectively. However, as the result of

peptide-induced outer membrane permeabilization, -lactamase can cleave NCF, which is

detected by a color change from yellow to red. The inner membrane permeabilization allows

ONPG to reach the cytoplasm, where -galactosidase metabolizes its substrate, which can be

followed by a color change from colorless to yellow (Fig. 41).

The inner and outer membrane permeabilizating activities of MEL, BUF and TP and

the AOA-modified peptides against E. coli (strain ML-35p) were determined in 96-well


92

microtiter plates. The bacterial culture was grown in LB medium containing 100 g/mL

ampicillin until an OD600 of 0.3 was reached; cells were then rinsed twice and resuspended in

HEPES buffer to an OD600 of 0.3. The microtiter wells were filled up with 50 L of the

dissolved peptides at individual concentrations close to the bacteria MIC. To assay inner

membrane permeabilization, 50 L of ONPG stock solution (300 g/mL) was added to the

microtiter wells and for monitoring the outer membrane permeabilization, 50 L of NCF

stock solution (60 g/mL) was pipetted into the wells. Finally, 50 L cell suspension

(OD600 of 0.3) was added to the wells and the absorbance was measured. The wells without

peptides served as controls. The inner and outer membrane permeabilizations were monitored

spectrophotometrically over a time period up to ~ 1 h at of 420 nm and 500 nm,

respectively.

6.2.6 Vesicle preparation

6.2.6.1 SUVs

The final concentrations of lipids for ITC experiments were 40 mM for POPC, 20 mM

for POPC mixed with POPG or LA and 5 mM for POPC mixed with r-LPS and s-LPS.

Synthetic lipids To make SUVs of different lipid mixtures, the dried lipid films, as prepared above,

were hydrated in pyrogen-free phosphate buffer (10 mM NaH2PO4/Na2HPO4, 154 mM NaF,

pH 7.4). The lipid concentration was calculated gravimetrically on the basis of amount of the

dried lipids used for the preparation of vesicles. The suspensions were vortexed for 5 min and

ultrasonicated in an ice/water bath using an ultrasonicator (Labsonic L, B. Braun Biotech,

Germany; and Sonopuls HD 2070, Bandelin electronic, Germany) with clamp for 20 min. The

size of the vesciles was determined by DLS on an N4 Plus particle sizer (Beckman Coulter,

U.S.A.) equipped with a 10-mW helium/neon laser with 632.8 nm at a scattering angle of

90. The mean diameter for SUVs was ~ 30 nm.

Natural lipids For the preparation of the POPC/LA (12/1 [mol/mol]) lipid film, dried LA (molecular

mass = 1792 Da.) was firstly dissolved in a volume of chloroform/methanol (2/1 [v/v]) to give

a concentration of 2.5 mg/ml. A certain volume of the dissolved LA was added to a 10 mL


93

round-bottomed glass tube prefilled with a specific amount of dried POPC lipid film to reach

the exact molar ratio of POPC/LA. Afterwards, the solvent was removed by rotary

evaporation, followed by the use of a high vacuum overnight in order to dry the uniform lipid

film.

To make the POPC/LPS (12/1 [mol/mol]) lipid film, we employed a protocol called

“dry method” [176]. According to this protocol, a certain amount of dry LPS was weighed

and added into a 10 mL round-bottomed glass tube, prefilled with specific amount of dried

POPC lipid film, such that the molar ratio of POPC/LPS reaches 12/1. Based on the structure

of LPS shown in Fig. 16, we assumed that Rd-LPS and s-LPS have molecular masses of

approximately 2.5 and 10 kDa., respectively. Because LPS is a heterogenous molecule and

has the tendency to aggregate in varying sizes, the exact value for the molecular mass of LPS

cannot be determined [177]. The assumed molecular masses for Rd-LPS and s-LPS used in

these studies fit well with the masses reported for Rd-LPS from Salmonella Minnesota and s-

LPS from E. coli (strain O8:K127) [177]. However; considering the overall heterogeneity, it

has been reported that the molecular mass for s-LPS from E. coli K-235 varies between

10−80 kDa. [178]. The dried POPC/LPS lipid was then suspended in pyrogen-free water

(Milli-Q, Element System, Millipore, France) and extensively vortexed. It was then heated up

to 60 C followed by sonication in a bath sonicator for 10 min. The suspension was vortexed

again and the cycle repeated twice. This turbid mixture was next lyophilized and made ready

for further application. This protocol leads to the maximum amount of LPS incorporated into

POPC phospholipid membrane [179]. To prevent any contamination of the POPC/LPS

vesicles, no further attempt was done to separate non-incorporated LPS.

6.2.6.2 LUVs

Appropriate amounts of POPC alone, and its mixture with POPG at molar ratios of 3:1

and 1:3, were prepared. The lipids films were perepared, as described above, for the synthetic

lipids. The lipid films were dried overnight under high vacuum and then suspended by vortex

mixing in calcein buffer solution at a self-quenching concentration (80 mM calcein,

10 mM TRIS, 0.1 mM EDTA, pH 7.4). Liposome size was reduced by extrusion

(Avestin Inc., Canada) 35 times through two stacked 100 nm pore size polycarbonate filters.

Untrapped calcein was removed from the LUVs by gel filtration on a Sephadex G50-

medium column (eluent: buffer containing 10 mM TRIS, 154 mM NaCl, 0.1 mM EDTA,


94

pH 7.4). A plastic syringe (5 mL volume, plugged with a filter pad) mounted in a

centrifugation tube was filled with hydrated Sephadex G-50 gel. After spinning at 2000 × g

for 5 min, the gel column had dried and could be parted from the sides of the syringe. 500 L

of the vesicle suspension was dropped onto the gel bed and the liposomes were eluted by

centrifugation at 2000 × g for 5 min. The collected liposomes were diluted 1:1 with buffer

containing salt and finally the lipid concentration was determined by phosphorus analysis

[44,118].

6.2.7 Lipid bilayer permeabizing activities

In the dye release assay, aliquots of the LUV suspensions, loaded with calcein at self-

quenching concentration were injected into cuvettes containing the dissolved peptides or

stirred suspensions of TentaGel S NH2-bound CAPs at different concentrations in buffer

(10 mM TRIS, 154 mM NaCl, 0.1 mM EDTA, pH 7.4). The lipid concentration was 25 M in

a total volume of 2 mL. The peptide-induced calcein release was monitored fluorimetrically

by measuring the time dependent decrease in self-quenching (excitation 490 nm,

emission 514 nm) at room temperature using a LS 50B spectrofluorimeter (Perkin-Elmer,

Germany) (Fig. 42). The fluorescence intensity corresponding to 100% release was

determined by addition of 100 L of a 10 % Triton X-100 solution. EC50s (or EC25s) were

derived from dose response curves giving the fluorescence intensity after 1 min.

Fig. 42. Schematic representation of CAPs-induced relase of liposome-loaded dye (calcein) measured by fluorescence.


95

6.2.8 Isothermal titration calorimetry

ITC is a useful technique to study chemical reactions quantitatively and describe them

thermodynamically. Each reaction is connected with an enthalpy change (�H°), which means

that heat is either released into (exothermic) or taken in from (endothermic) the surroundings.

This universal property is independent of molecule size and so that all chemical and

biochemical reactions can be studied [180]. This includes the formation of complexes

between proteins, DNA, lipids or low molecular weight biomolecules, which are mostly based

on non-covalent bonds. A particular example is the interaction between peptides (P) and lipid

(L) bilayers mimicking bacterial membranes [181]. The complex formation (L.P) can be

described by the simple equilibrium shown below:

L + P L.P�

This equilibrium is defined by a binding constant (K) as given by:

[L.P]=

[L][P]K (1).

In Fig. 43, the operation of an isothermal titration calorimeter is schematically shown.

It consists of a reference and a sample cell. The temperature difference between two cells can

be very precisely measured. The ITC syringe is filled with a solution of the interaction partner

L (liposomes) and a solution of binding partner P (peptide) is present in the sample cell. The

reference cell is filled with buffer. It serves as the reference for the temperature change, which

takes place in the sample cell. Both cells have a higher temperature than the carefully

thermostated environment (hold isothermal) and are supplied with controlled and measurable

heating to keep the temperature constant. Each injection from the syringe leads to the

formation of the L.P complex and this process is accompanied by heat absorption/release

from/to the sample cell. The calorimeter responds to the endothermic or exothermic reaction

with a corresponding increase or decrease of the electrical heating power to keep the

temperature in the sample cell constant. These responses appear as differential heating power,

as shown e.g. for heat release in the upper part of Fig. 43B. By integrating the area under each

signal, the free-set quantity of heat versus the increasing ratio of L and P is obtained (lower

part of Fig. 43B). By adjusting the theoretical curve to these data, K is obtained.


96

Fig. 43.(A) Scheme of an isothermal titration calorimeter, and (B) a typical ITC experiment. The red and blue arrows (shown left) represent the exactly equilibrated heat flow from the electric heater (red) to the reference and the sample cell and from there to the cooler surroundings (blue). The smallest changes in

temperature (∆T) between the two cells are detected and compensated by changing the heating power. In the right panel, the change in heating power for each injection into the sample cell is presented as a trace of negative

peaks, which become smaller by increasing the number of injection because less substance is available for binding.

ITC can provide the comprehensive thermodynamic characterization of binding of

antimicrobial peptides to different lipid vesicles [181]. The thermodynamic parameters in

terms of ∆H°, ∆S°, and ∆G° are useful to understand the mechanism of action of CAPs and

their selectivity towards different lipid membranes.

6.2.8.1 Theory and description of surface partitioning equilibrium model

The interaction between a peptide and a lipid membrane surface can be described by a

two-step model. The interaction is initiated by electrostatic contributions resulting in surface

accumulation. This is followed by hydrophobic interactions causing peptide insertion [181].

In order to describe the peptide-lipid binding, a surface partitioning equilibrium model was

used to fit all ITC experimental data and draw the thermodynamic parameters [182,183].

Based on this model, a non-ideal mixing was assumed in both the aqueous solution

and the bilayer phase in order to describe the peptide partitioning into the lipid surface

adequately (see ref. 182, and 183). Peptide binding from the bulk aqueous solution to the lipid

bilayer, so-called Kapp., includes both hydrophobic and electrostatic contributions for peptide-

lipid interaction and is defined as:


97

bapp.

P,f

=R

Kc

(2),

where Rb is:

P,bb

L

=c

Rγ c

(3).

γ was taken as 0.6 for membrane-impermeant peptides interacting with SUVs [181]. Rb was

assumed to be given by:

eb 0 P,i 0 P,f exp

ϕ− ∆ = =

z eR K c K c

kT (4),

where e, k, and T are the elementary charge (1.602×10-9 C), the Boltzmann constant, and the

absolute temperature, respectively. According to the Gouy–Chapman theory, ∆ϕ is related to

the membrane surface charge density, as described by Seelig and Keller et al (Eq. 4)

[181−183].

0 r2

i 0i2000 [ ( ) 1]ϕ∆∑i

σ = ε ε RT c exp -z F RT - (5),

where zi is the valence of ith species, and ci is the concentration of the ith dissociated

electrolyte in the solution. σ is obtained according to the theoretical calculation described by

Seelig [184], which is corrected for the effect of counterion binding due to the association of

Na+ with the POPG headgroups as given by:

( )( )P b

0L b P Na Na1 exp( )ϕ∆z eR

σ =A + R A + K c F RT

(6),

where AL is 68 nm2 and AP is assumed zero when the peptide adsorbs superficially. KNa+ is the

binding constant of the sodium to the negatively charged lipid headgroup (0.6 M-1) with a

concentration of cNa+.

In an ITC experiment, the normalized heat produced or consumed on peptide-lipid

interactions (Q) is given by (see ref. 182 for details):

P,b P,b P,s dil.L

∆ ∆ ∆ º= - 1- - +

∆

cell cellcell

cell cell

V V HQ V c ć c Q

V V n (7),


98

where ∆H° stands for the molar transfer enthalpy of the peptide from the aqueous into the

bilayer phase, and Qdil, for the heat of dilution normalized with respect to the molar amount of

lipid injected, ∆nL. ćP,b and cP,b are considered as the equilibrium concentrations of bound

peptide before and after an injection of volume ∆Vcell, respectively, and cP,s that in the syringe.

On the basis of this model, we fitted the ITC data to obtain K0, ∆H°, and ze for each

peptide. The ∆G° was then taken as:

0∆ = - ln(55.5 M )°G RT K (8),

where 55.5 M is the molar concentration of water in the aqueous phase [185]. Finally, the

entropic contribution to membrane partitioning, −T∆S°, was calculated from the Gibbs–

Helmholtz equation as

- ∆ ° = ∆ ° -∆ °T S G H (9).

6.2.8.2 Instrument setup and measurement

High-sensitivity ITC was performed on a VP-ITC (GE Healthcare, Sweden). All

experiments were run at 37 °C to study peptide binding to POPC doped with LA and LPSs

above the gel-to-liquid-crystalline phase transition temperature. At lower temperatures, the

binding process is endothermic [186]. To determine peptide-lipid interactions, a 40 µM

peptide solution in the calorimeter cell was titrated with an SUV suspension ranging from 5 to

40 mM. All peptide solutions were degassed before ITC experiments. In a typical experiment,

3−10 µL aliquots of the SUV suspension were titrated into the peptide solution during each

injection and the content of the sample cell was stirred continuously at 310 rpm. Because of a

small loss of titrant during the mounting of the syringe and the equilibration stage preceding

the actual titration, the first injection volume was usually set to 3−5 µL. The first injection

peak was excluded during data analysis. Time spacings of 5 min were long enough to allow

the ITC signal to return to the baseline value. The measured heat of binding decreased with

consecutive lipid injections because less free peptide was available. Control experiments,

injecting lipid vesicles into buffer without peptide, showed that the heats of dilution were

small and constant.


99

6.2.8.3 ITC data analysis and curve fitting

The raw ITC data were collected based on the program supplied by the manufacturer.

Baseline correction, peak integration and its adjustment for data acquisition and analysis were

conducted as described by the manufacturer. Nonlinear least-squares data fitting was

performed in an Excel (Microsoft, U.S.A.) spreadsheet using the Solver add-in (Frontline

Systems, U.S.A.) [187].

6.2.9 CD spectroscopy

CD spectroscopy is a technique by which conformational properties of peptides can be

quickly measured in solution. CD occurs due to the difference in absorption of left or right

circularly polarized light in the UV−VIS wavelength range (100−1000 nm) by optically active

chromophores. In addition to inherent chiral absorbing systems, achiral chromophores also

show a CD when they are disturbed by the dissymmetric field of an optically active group. In

the absence of aromatic amino acid residues, only the peptide backbone contributes

significantly in the far UV region. Thus, the CD spectra of peptides are determined by

transition dipole moments of the amide groups, which are influenced by the chiral Cα-atoms.

Absorption occurs mainly in the range between 180−240 nm: π−π* transitions (λ < 200 nm)

and n−π* transitions (λ = ~ 218 nm) with less intensity. The superposition of contributions

from aromatic residues in the 220 to 240 nm region influences the spectral characteristics. The

induction of a secondary structure confers a structural asymmetry upon peptide sequences,

which is reflected by characteristic CD patterns. There are three main classes of secondary

structures: the α-helix, the β-sheet, and the random coil. The α-helix produces the most

distinctive CD spectrum: a very strong positive band near 192 nm, which corresponds to

π−π*⊥ transitions and two negative maxima of approximately equal intensity near 222 nm

(n−π*) and 208 nm (π−π*||). The β-sheet exhibits a single negative band near 217 nm,

representing n−π*. The random coil exhibits a strong negative band at 197 nm (π−π*||) and a

small positive band in the 220 nm (n−π*) region. In the spectra of complex proteins, the

contributions of the individual structures superimpose. The spectra provide less information

about the conformation of certain parts of the molecule, but rather provide an integrated

picture of a peptide’s structure. The value of the method is in the sensitivity of the CD signal

with respect to structural changes induced by variations in the peptide concentration, pH,

change in temperature, solvent polarity or ionic additives [188]. However, based upon the


100

helicity of an ideal helix, or the correlation of CD spectra of proteins and their known crystal

structure, also the content of helix or amount of other structures of peptides and proteins of

interest can be calculated. However, these methods are only slightly suitable for small

peptides because small sequences form poor helices and the data evaluation is based on the

analysis of proteins with secondary and tertiary structures [189]. In general, the CD spectrum

of a peptide represents the Θmr versus λ.

6.2.9.1 c-WFW and W-subtituted analogs

Stock solutions of cyclic peptides (2.5 mM) were prepared by dissolving the samples

in buffer (10 mM phosphate buffer, 154 mM NaF, pH 7.4). CD measurements were

performed in far-UV (190−260 nm) and near-UV (240−340 nm) regions. The measurements

were conducted in phosphate buffer, buffer/TFE (1/1 [v/v]), buffer/GnHCl (1/1 [v/v]), SDS-

and POPG-bound peptides at the desired tempretaure. Thus, an aliquot of the peptide stock

solution was diluted with: (i) the phosphate buffer, (ii) TFE, (iii) 8M GnHCl, (iv)

50 mM suspension of SDS and (v) 20 mM suspension of POPG SUVs in the same buffer to

achieve the desired peptide (100 µM), GnHCl (4 M), SDS (25 mM), and POPG (10 mM)

concentrations. Measurements were carried out on a Jasco J-720 spectropolarimeter (Jasco,

Japan) at either 20 °C or 80 °C. To increase the signal/noise ratio, 35 scans were accumulated.

Peptide spectra were corrected by subtracting spectra of the corresponding buffer or peptide-

free lipid suspensions. The presented spectra give Θmr.

6.2.9.2 Model KLAL peptide, MK5E, and the PEGylated analogs

200 µM stock peptide solutions were prepared by dissolving the samples in buffer

(10 mM phosphate buffer, 154 mM NaF, pH 7.4). The solutions were mixed 1/1 (v/v) with

TFE to get the desired peptide concentration (50 µM) and solvent composition.

CD measurements were carried out on a J-720 spectropolarimeter over a λ = 185−260 nm at

room temperature. Twenty CD scans were accumulated for each sample. The helicity was

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.

Appendix

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8 Appendix

8.1 Curriculum vitae

For reasons of data protection, the curriculum vita is not included in the online version.

8.2 Publications and scientific conference contributions

8.2.1 Original publications

Articles in journals

Bagheri, M., M. Beyermann, and M. Dathe. 2010. Membrane-active peptides:

tethering on solid surfaces to get information on the mode of action. Eur. Biophys. J.

In Press.

Bagheri, M., S. Keller, and M. Dathe. 2010. Interaction of W-substituted analogs of

cyclo-RRRWFW with bacterial lipopolysaccharides: The role of the aromatic cluster

in antimicrobial activity. Antimicrob. Agents Chemother. In press.

Junkes, C., R. Harvey, K. D. Bruce, R. Dölling, M. Bagheri, and M. Dathe. 2010.

Cyclic antimicrobial R-, W-rich peptides: structure and membrane composition

determine the mechanism of action. Eur. Biophys. J. In Press.

Bagheri, M. 2010. Synthesis and thermodynamic characterization of small cyclic

antimicrobial arginine and tryptophan-rich peptides with selectivity for Gram-negative

bacteria, Vol. 618. p. 87−109. In A. Giuliani and A. C. Rinaldi (eds.), Antimicrobial

peptides: methods and protocols, methods in molecular biology. Humana press, New

York.

Bagheri, M., M. Beyermann, and M. Dathe. 2009. Immobilization reduces the activity

of surface-bound cationic antimicrobial peptides with no influence upon the activity

spectrum. Antimicrob. Agents Chemother. 53:1132−1141.

Bagheri, M., N. Azizi, and M. R. Saidi. 2005. An intriguing effect of lithium

perchlorate dispersed on silica gel in the bromination of aromatic compounds by N-

bromosuccinimide. Can. J. Chem. 83:146−149.

Matloubi-Moghaddam, F., H. Zali-Boeini, M. Bagheri, P. Rüedi, A. Linden. 2005.

Highly efficient and versatile one-pot synthesis of new thiophenelyidene compounds.

J. Sulfur Chem. 26:245−250.

Appendix

120

Published contributions to academic conferences

Bagheri, M., S. Keller, and M. Dathe. 2010. Interactions of W-substituted cyclo-

RRRWFW analogs with lipid membranes studied by isothermal titration calorimetry.

Proceedings of the 12th Iberian peptide meeting at Lisbon, University of Lisbon.

February 2010, pp. 50−50 (in Portugal).

Bagheri, M., M. Beyermann, and M. Dathe. 2008. Surface-bound cationic

antimicrobial peptides: the effect of immobilization upon the activity spectrum and the

mode of action. Proceedings of the 30th European Peptide Symposium (Peptides 2008)

at Helsinki, ONGREX / Blue & White Conferences Oy. September 2008, pp. 208−209

(in Finland).

8.2.2 Scientific conference contributions

“The role of tryptophan in activity and E. coli selectivity of cyclic hexapeptides”

Poster at the 455th WE-Heraeus-Seminar on Biophysics of Membrane-Active

Peptides. Physikzentrum Bad Honnef, Bad Honnef, Germany, April 11th−April 14th,

2010.

“Interactions of W-substituted cyclo-RRRWFW analogs with lipid membranes studied

by isothermal titration calorimetry” Oral Presentation at 12th Iberian peptide

meeting. The Faculty of Medicine, University of Lisbon, Lisbon, Portugal, 10th – 12th

February 2010.

“Surface-bound cationic antimicrobial peptides: The effect of immobilization upon

the activity spectrum and the mode of action” Oral Presentation at 30th European

Peptide Symposium (30EPS)−Young Investigators Mini Symposium.

ONGREX / Blue & White Conferences Oy, Helsinki, Finland,

August 31st−September 5th, 2008.

“Polymer-bound Magainin-derived MK5E and KLAL; The effect of spacer length on

the antimicrobial activities” Poster at the 6th Gordon Research Conferences (GRC)

on Antimicrobial Peptides. Il Ciocco, Lucca (Barga), Italy, April 29th−May 4th, 2007.

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